SERPINC1 iRNA COMPOSITIONS AND METHODS OF USE THEREOF

ABSTRACT

The invention relates to iRNA, e.g., double-stranded ribonucleic acid (dsRNA), compositions targeting the Serpinc1 gene, and methods of using such iRNA, e.g., dsRNA, compositions to inhibit expression of Serpinc1 and methods of treating subjects having a bleeding disorder, such as a hemophilia.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/837,129, filed Mar. 15, 2013, which claims priority to U.S.Provisional Application No. 61/638,952, filed on Apr. 26, 2012, to U.S.Provisional Application No. 61/669,249, filed on Jul. 9, 2012, and toU.S. Provisional Application No. 61/734,573, filed on Dec. 7, 2012. Theentire contents of each of the foregoing applications are incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 21, 2015, isnamed SeqList_(—)00205.txt and is 458,189 bytes in size.

BACKGROUND OF THE INVENTION

Serpinc1 is a member of the serine proteinase inhibitor (serpin)superfamily. Serpinc1 is a plasma protease inhibitor that inhibitsthrombin as well as other activated serine proteases of the coagulationsystem, such as factors X, IX, XI, XII and VII and, thus, regulates theblood coagulation cascade (see, e.g., FIG. 1). The anticoagulantactivity of Serpinc1 is enhanced by the presence of heparin and otherrelated glycosaminoglycans which catalyze the formation of athrombin:antithrombin (TAT) complexes.

Bleeding disorders, either inherited or acquired, are conditions inwhich there is inadequate blood clotting. For example, hemophilia is agroup of hereditary genetic bleeding disorders that impair the body'sability to control blood clotting or coagulation. Hemophilia A is arecessive X-linked genetic disorder involving a lack of functionalclotting Factor VIII and represents 80% of hemophilia cases. HemophiliaB is a recessive X-linked genetic disorder involving a lack offunctional clotting Factor IX. It comprises approximately 20% ofhaemophilia cases. Hemophilia C is an autosomal genetic disorderinvolving a lack of functional clotting Factor XI. Hemophilia C is notcompletely recessive, as heterozygous individuals also show increasedbleeding.

Although, at present there is no cure for hemophilia, it can becontrolled with regular infusions of the deficient clotting factor,e.g., factor VIII in hemophilia A. However, some hemophiliacs developantibodies (inhibitors) against the replacement factors given to themand, thus, become refractory to replacement coagulation factor.Accordingly, bleeds in such subjects cannot be properly controlled.

The development of high-titer inhibitors to, for example, factor VIIIand other coagulation factors, is the most serious complication ofhemophilia therapy and makes treatment of bleeds very challenging.Currently, the only strategies to stop bleeds in such subjects are theuse of “bypassing agents” such as factor eight inhibitor bypass activity(FEIBA) and activated recombinant factor VII (rFVIIa), plasmapheresis,continuous factor replacement, and immune tolerance therapy, none ofwhich are completely effective. Accordingly, there is a need in the artfor alternative treatments for subjects having a bleeding disorder, suchas hemophilia.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of a Serpinc1 gene. The Serpinc1 gene may be within a cell,e.g., a cell within a subject, such as a human. The present inventionalso provides methods of using and uses of the iRNA compositions of theinvention for inhibiting the expression of a Serpinc1 gene and/or fortreating a subject having a disorder that would benefit from inhibitingor reducing the expression of a Serpinc1 gene, e.g., a bleedingdisorder, such as hemophilia.

Accordingly, in one aspect, the present invention providesdouble-stranded ribonucleic acids (dsRNAs) for inhibiting expression ofSerpinc1. The dsRNAs comprise a sense strand and an antisense strand,wherein the sense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from the nucleotide sequence ofSEQ ID NO:1 and the antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequence of SEQ ID NO:5.

In another aspect, the present invention provides double-strandedribonucleic acids (dsRNAs) for inhibiting expression of Serpinc1. ThedsRNAs comprise a sense strand and an antisense strand, the antisensestrand comprising a region of complementarity which comprises at least15 contiguous nucleotides differing by no more than 3 nucleotides fromany one of the antisense sequences listed in any one of Tables 3, 4, 8,11, 12, 14, 15, 20, and 21.

In one embodiment, the sense and antisense strands comprise sequencesselected from the group consisting of AD-50487.1, AD-50477.1,AD-50483.1, AD-50475.1, AD-50495.1, AD-50476.1, AD-50499.1, AD-50478.1,AD-50489.1, AD-50501.1, AD-50507.1, AD-50484.1, AD-50515.1, AD-50540.1,AD-50528.1, AD-50549.1, AD-50539.1, AD-50534.1, AD-50527.1, AD-50514.1,AD-50509.1, AD-50529.1, AD-54944, AD-56813, AD-57205, AD-57214, andAD-57213 of any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21. Incertain embodiments of the invention, the dsRNAs comprise at least onemodified nucleotide. In one embodiment, at least one of the modifiednucleotides is selected from the group consisting of a 2′-O-methylmodified nucleotide, a nucleotide comprising a 5′-phosphorothioategroup, and a terminal nucleotide linked to a cholesteryl derivative or adodecanoic acid bisdecylamide group. In another embodiment, the modifiednucleotide is selected from the group consisting of a 2′-deoxy-2′-fluoromodified nucleotide, a 2′-deoxy-modified nucleotide, a lockednucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a2′-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide.

The region of complementarity of the dsRNAs may be at least 17nucleotides in length, between 19 and 21 nucleotides in length, or 19nucleotides in length.

In one embodiment, each strand of a dsRNA is no more than 30 nucleotidesin length.

At least one strand of a dsRNA may comprise a 3′ overhang of at least 1nucleotide or at least 2 nucleotides.

In certain embodiments, a dsRNA further comprises a ligand. In oneembodiment, the ligand is conjugated to the 3′ end of the sense strandof the dsRNA.

In some embodiments, the ligand is one or more N-acetylgalactosamine(GalNAc) derivatives attached through a bivalent or trivalent branchedlinker. In particular embodiments, the ligand is

In some embodiments, the RNAi agent is conjugated to the ligand as shownin the following schematic

In another embodiment, the RNAi agent is conjugated to the ligand asshown in the following schematic, wherein X is O or S.

In one embodiment, the region of complementarity of a dsRNA consists ofone of the antisense sequences of any one of Tables 3, 4, 8, 11, 12, 14,15, 20, and 21.

In another embodiment, a dsRNA comprises a sense strand consisting of asense strand sequence selected from the sequences of any one of Tables3, 4, 8, 11, 12, 14, 15, 20, and 21, and an antisense strand consistingof an antisense sequence selected from the sequences of any one ofTables 3, 4, 8, 11, 12, 14, 15, 20, and 21.

In another aspect, the present invention provides a cell containing adsRNA of the invention.

In yet another aspect, the present invention provides a vector encodingat least one strand of a dsRNA, wherein the dsRNA comprises a region ofcomplementarity to at least a part of an mRNA encoding Serpinc1, whereinthe dsRNA is 30 base pairs or less in length, and wherein the dsRNAtargets the mRNA for cleavage.

The region of complementarity may be least 15 nucleotides in length or19 to 21 nucleotides in length.

In a further aspect, the present invention provides a cell comprising avector encoding at least one strand of a dsRNA, wherein the dsRNAcomprises a region of complementarity to at least a part of an mRNAencoding Serpinc1, wherein the dsRNA is 30 base pairs or less in length,and wherein the dsRNA targets the mRNA for cleavage.

In one aspect, the present invention provides a pharmaceuticalcomposition for inhibiting expression of a Serpinc1 gene comprising adsRNA or vector of the invention.

In one embodiment, the pharmaceutical composition further comprises alipid formulation, such as an MC3, SNALP, or XTC formulation.

In another aspect, the present invention provides methods of inhibitingSerpinc1 expression in a cell. The methods include contacting the cellwith the dsRNA or a vector of the invention, and maintaining the cellproduced for a time sufficient to obtain degradation of the mRNAtranscript of a Serpinc1 gene, thereby inhibiting expression of theSerpinc1 gene in the cell.

The cell may be within a subject, such as a human subject, for example ahuman subject suffering from a bleeding disorder, e.g., a hemophilia.

In one embodiment of the methods of the invention, Serpinc1 expressionis inhibited by at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99%.

In another aspect, the present invention provides methods of treating asubject having a disorder that would benefit from reduction in Serpinc1expression, e.g., a bleeding disorder, such as a hemophilia. The methodsinclude administering to the subject a therapeutically effective amountof the dsRNA or vector of the invention, thereby treating the subject.

In one aspect, the invention provides methods of preventing at least onesymptom, e.g., bleeding, in a subject having a disorder that wouldbenefit from reduction in Serpinc1 expression, e.g., hemophilia. Themethods include administering to the subject a therapeutically effectiveamount of the RNA, e.g., dsRNA, or vector of the invention, therebypreventing at least one symptom in the subject having a disorder thatwould benefit from reduction in Serpinc1 expression.

The disorder may be a bleeding disorder, such as a hemophilia.

In one embodiment, the administration of the dsRNA to the subject causesan increase in blood clotting and/or a decrease in Serpinc1 proteinexpression and/or accumulation.

In one embodiment, the dsRNA is conjugated to a ligand, e.g., at the3′-end of the sense strand of the dsRNA. In one embodiment the ligand isan N-acetylgalactosamine (GalNAc) derivative.

In one embodiment, the dsRNA is administered at a dose of about 0.01mg/kg to about 10 mg/kg, e.g., about 0.05 mg/kg to about 5 mg/kg, about0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kgto about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg toabout 10 mg/kg, about 2 mg/kg to about 2.5 mg/kg, about 2 mg/kg to about10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg,about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5.5mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5mg/kg to about 10 mg/kg. Values and ranges intermediate to the recitedvalues are also intended to be part of this invention.

For example, the dsRNA may be administered at a dose of about 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

In another embodiment, the dsRNA is administered at a dose of about 0.5to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

For example, subjects can be administered a therapeutic amount of iRNA,such as about 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5,13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5,20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5,27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention.

The dsRNA, e.g., conjugated to a ligand, may be administered to thesubject once a week or twice a month.

In another aspect, the present invention provides methods of inhibitingthe expression of Serpinc1 in a subject. The methods includeadministering to the subject a therapeutically effective amount of thedsRNA or a vector of the invention, thereby inhibiting the expression ofSerpinc1 in the subject.

In one embodiment, the dsRNA is conjugated to a ligand, e.g., at the3′-end of the sense strand of the dsRNA. In one embodiment the ligand isan N-acetylgalactosamine (GalNAc) derivative.

In one embodiment, the dsRNA is administered at a dose of about 0.01mg/kg to about 10 mg/kg, e.g., about 0.05 mg/kg to about 5 mg/kg, about0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kgto about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg toabout 10 mg/kg, about 2 mg/kg to about 2.5 mg/kg, about 2 mg/kg to about10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg,about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5.5mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5mg/kg to about 10 mg/kg. Values and ranges intermediate to the recitedvalues are also intended to be part of this invention.

For example, the dsRNA may be administered at a dose of about 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

In another embodiment, the dsRNA is administered at a dose of about 0.5to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

For example, subjects can be administered a therapeutic amount of iRNA,such as about 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5,13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5,20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5,27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention.

The dsRNA, e.g., conjugated to a ligand, may be administered to thesubject once a week or twice a month.

In yet another aspect, the invention provides kits for performing themethods of the invention. In one embodiment, the invention provides akit for performing a method of inhibiting expression of Serpinc1 in acell by contacting a cell with a double stranded RNAi agent in an amounteffective to inhibit expression of the Serpinc1 gene in the cell. Thekit comprises an RNAi agent and instructions for use and, optionally,means for administering the RNAi agent to a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the blood coagulation cascade.

FIGS. 2A and 2B are graphs showing the inhibition of Serpinc1 expressionin Hep3B cells following a single dose of the indicated iRNAs.

FIGS. 3A and 3B are graphs showing the inhibition of Serpinc1 mRNA (A)and protein (B) expression in CD-1 mice following a single dose, asindicated, of an LNP formulation of AD-50509 or AD-1955.

FIGS. 4A and 4B are graphs showing the duration of inhibition ofSerpinc1 mRNA (A) and protein (B) expression in CD-1 mice following asingle 1 mg/kg dose of an LNP formulation of AD-50509 or AD-1955. FIG.4C is a graph showing the inhibition of Serpinc1 activity and Serpinc1protein expression in CD1 mice following a single 1 mg/kg dose of an LNPformulation of AD-50509 or AD-1955.

FIG. 5 is a graph showing the percent knock-down of Serpinc1 mRNA andprotein levels following a single 10 mg/kg dose of the indicated iRNAconjugated to GalNAc.

FIG. 6 is a graph showing the inhibition of Serpinc1 protein expressionin C57BL/6 mice following a single 5 mg/kg, 10 mg/kg, 25 mg/kg, 50mg/kg, and 75 mg/kg, and a repeat dose of 5 X 5 mg/kg of AD-54944conjugated to GalNAc.

FIGS. 7A and 7B are graphs showing the effect of repeat-dosing on theduration of inhibition of Serpinc1 protein expression in C57BL/6 mice ofGalNAc conjugated AD-54944.

FIGS. 8 and 9 are graphs showing the effects of the indicatedsplit-dosing regimens on the duration of silencing of Serpinc1 proteinexpression in C57BL/6 mice administered GalNAc conjugated AD-54944.

FIG. 10A is a graph showing the percent knock-down of Serpinc1 proteinlevels following a single 10 mg/kg dose of the indicated iRNA conjugatedto GalNAc.

FIG. 10B is a graph showing the percent knock-down of Serpinc1 proteinlevels following a single 3 mg/kg dose of the indicated iRNA conjugatedto GalNAc.

FIG. 11 is a graph showing the percent knock-down of Serpinc1 proteinlevels following a single 10 mg/kg or 3 mg/kg dose of the indicated iRNAconjugated to GalNAc.

FIG. 12 is a graph showing the percent knock-down of Serpinc1 activityfollowing a single 10 mg/kg or 3 mg/kg dose of the indicated iRNAconjugated to GalNAc.

FIG. 13 is a graph showing a dose effect response to a single dose ofAD-57213.

FIG. 14 is a graph showing the duration of silencing of Serpinc1 withAD-57213 following a single dose of 1 mg/kg, 3 mg/kg or 10 mg/kg inHemophilia A mice.

FIG. 15 is a graph showing the inhibition of Serpinc1 mRNA expression inC57BL/6 mice following a single 30 mg/kg, 10 mg/kg, 3 mg/kg, 1 mg/kg,and 0.3 mg/kg dose of AD-57213.

FIG. 16A is a graph showing the duration of silencing of Serpinc1 withAD-57213 following a single dose as indicated.

FIG. 16B is a graph showing the duration of silencing of Serpinc1 withAD-57205 following a single dose as indicated.

FIG. 16C is a graph showing the duration of silencing of Serpinc1 withAD-57214 following a single dose as indicated.

FIGS. 17-19 are graphs showing the effects of the indicated split-dosingregimens on the duration of silencing of Serpinc1 protein expression inC57BL/6 mice administered GalNAc conjugated AD-57213.

FIG. 20 is a graph showing the effects of the single dose screen of theindicated compounds on the duration of Serpinc1 protein expression innon-human primates.

FIG. 21 is a graph showing the effects of the single dose screen ofAD-57213 conjugated to GalNAc on the duration of Serpinc1 proteinexpression in non-human primates.

FIG. 22 is a graph showing the effects of the single dose screen of theindicated compounds on the duration of Serpinc1 protein expression innon-human primates.

FIG. 23 is a graph showing the effects of the single dose of compoundAD-57213 on serum antithrombin (Serpinc1) levels in non-human primates.

FIGS. 24A-24D are graphs showing the effects of the single dose ofcompound AD-57213 at A) 1 mg/kg, B) 3 mg/kg, C) 10 mg/kg, and D) 30mg/kg on the relationship between serum antithrombin (Serpinc1) levelsand fold change in peak plasma thrombin levels in non-human primates.Fold change in peak thrombin is depicted on the secondary y-axis (grey)and relative antithrombin level is depicted on the primary y-axis(black).

FIG. 25 is a graph showing the effects of AD-57213 as a fold changeincrease in peak thrombin as a function of relative antithrombin(Serpinc1) silencing.

FIG. 26A is a graph showing the cumulative effects of Serpinc1 silencingin non-human primates.

FIG. 26B is a graph showing the cumulative effects of Serpinc1 silencingin non-human primates.

FIGS. 27A and 27B are graphs showing the effects of a multi-doseadministration (0.5 mg/kg qw, 1 mg/kg q2w, 1.5 mg/kg qw, 3 mg/kg q2w) ofa Serpinc1 siRNA on serum antithrombin levels in non-human primates.Data points represent group mean, error bars represent standarddeviation (N=3). (qw=weekly; q2w=every other week).

FIG. 28A is a graph showing the effect of Serpinc1 silencing on plateletaccumulation following microvessel laser injury. The graph shows themedian values from all inflicted injuries FIG. 28B is a graph showingthe effect of Serpinc1 silencing on fibrin area following microvessellaser injury. The graph shows the median values from all inflictedinjuries.

FIG. 29 is a graph showing the duration of silencing of Serpinc1following administration of compound AD-57213 formulated in a lipidnucleic acid particle.

FIG. 30A shows the nucleotide sequence of Homo sapiens serpin peptidaseinhibitor, Glade C (antithrombin), member 1 (SERPINC1) (SEQ ID NO:1);FIG. 30B shows the nucleotide sequence of Macaca mulatta serpinpeptidase inhibitor, Glade C (antithrombin), member 1 (SERPINC1) (SEQ IDNO:2); FIG. 30C shows the nucleotide sequence of Mus musculus serine (orcysteine) peptidase inhibitor, Glade C (antithrombin), member 1(Serpinc1) (SEQ ID NO:3); FIG. 30D shows the nucleotide sequence ofRattus norvegicus serpin peptidase inhibitor, Glade C (antithrombin),member 1 (Serpinc1) (SEQ ID NO:4); FIG. 30E shows the reverse complementof SEQ ID NO:1 (SEQ ID NO:5); FIG. 30F shows the reverse complement ofSEQ ID NO:2 (SEQ ID NO:6); FIG. 30G shows the reverse complement of SEQID NO:3 (SEQ ID NO:7); FIG. 30H shows the reverse complement of SEQ IDNO:4 (SEQ ID NO:8); and FIG. 30I shows the amino acid sequence of anexemplary hydrophobic MTS-containing peptide, RFGF (SEQ ID NO: 9); theamino acid sequence of an exemplary RFGF analogue (SEQ ID NO: 10); theamino acid sequence of the HIV Tat protein (SEQ ID NO: 11); the aminoacid sequence of the Drosophila Antennapedia protein (SEQ ID NO: 12);and the amino acid sequence of an exemplary Peptide-based CleavableLinking Group.

FIG. 31A is a graph depicting that antithrombin reduction increasesthrombin generation in Factor IX-depleted human plasma in vitro.

FIG. 31B is a graph depicting that antithrombin reduction increasesthrombin generation in Factor IX-depleted human plasma in vitro.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of a Serpinc1 gene. The Serpinc1 gene may be within a cell,e.g., a cell within a subject, such as a human. The present inventionalso provides methods of using the iRNA compositions of the inventionfor inhibiting the expression of a Serpinc1 gene and/or for treating asubject having a disorder that would benefit from inhibiting or reducingthe expression of a Serpinc1 gene, e.g., a bleeding disorder, such ashemophilia. The present invention further provides methods forpreventing at least one symptom, e.g., bleeding, in a subject having adisorder that would benefit from inhibiting or reducing the expressionof a Serpinc1 gene, e.g., a bleeding disorder, such as hemophilia.

The iRNAs of the invention include an RNA strand (the antisense strand)having a region which is about 30 nucleotides or less in length, e.g.,15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21,15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25,18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26,19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27,20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27,21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which regionis substantially complementary to at least part of an mRNA transcript ofa Serpinc1 gene. The use of these iRNAs enables the targeted degradationof mRNAs of a Serpinc1 gene in mammals. Very low dosages of Serpinc1iRNAs, in particular, can specifically and efficiently mediate RNAinterference (RNAi), resulting in significant inhibition of expressionof a Serpinc1 gene. The present inventors have demonstrated that iRNAstargeting Serpinc1 can mediate RNAi in vitro and in vivo, resulting insignificant inhibition of expression of a Serpinc1 gene. Thus, methodsand compositions including these iRNAs are useful for treating a subjectwho would benefit by a reduction in the levels and/or activity of aSerpinc1 protein, such as a subject having a bleeding disorder, e.g.,hemophilia.

The following detailed description discloses how to make and usecompositions containing iRNAs to inhibit the expression of a Serpinc1gene, as well as compositions, uses, and methods for treating subjectshaving diseases and disorders that would benefit from inhibition and/orreduction of the expression of this gene.

I. DEFINITIONS

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

As used herein, “Serpinc1” refers to a particular polypeptide expressedin a cell. Serpinc1 is also known as serpin peptidase inhibitor, Glade C(antithrombin), member 1; antithrombin III; AT3; antithrombin; andheparin cofactor 1. The sequence of a human Serpinc1 mRNA transcript canbe found at, for example, GenBank Accession No. GI:254588059(NM_(—)000488; SEQ ID NO:1). The sequence of rhesus Serpinc1 mRNA can befound at, for example, GenBank Accession No. GI:157167169(NM_(—)001104583; SEQ ID NO:2). The sequence of mouse Serpinc1 mRNA canbe found at, for example, GenBank Accession No. GI:237874216(NM_(—)080844; SEQ ID NO:3). The sequence of rat Serpinc1 mRNA can befound at, for example, GenBank Accession No. GI:58865629(NM_(—)001012027; SEQ ID NO:4). The term“Serpinc1” as used herein alsorefers to a particular polypeptide expressed in a cell by naturallyoccurring DNA sequence variations of the Serpinc1 gene, such as a singlenucleotide polymorphism in the Serpinc1 gene. Numerous SNPs within theSerpinc1 gene have been identified and may be found at, for example,NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov/sup). Non-limiting examplesof SNPs within the Serpinc1 gene may be found at, NCBI dbSNP AccessionNos. rs677; rs5877; rs5878; rs5879; rs941988; rs941989; rs1799876;rs19637711; rs2008946; and rs2227586.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a Serpinc1 gene, including mRNA that is a product of RNA processingof a primary transcription product. In one embodiment, the targetportion of the sequence will be at least long enough to serve as asubstrate for iRNA-directed cleavage at or near that portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a Serpinc1 gene.

The target sequence may be from about 9-36 nucleotides in length, e.g.,about 15-30 nucleotides in length. For example, the target sequence canbe from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of theinvention.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacement moiety(see, e.g., Table 2). The skilled person is well aware that guanine,cytosine, adenine, and uracil can be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base can basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent”as used interchangeably herein, refer to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.iRNA directs the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi). The iRNA modulates, e.g., inhibits,the expression of Serpinc1 in a cell, e.g., a cell within a subject,such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a singlestranded RNA that interacts with a target RNA sequence, e.g., a Serpinc1target mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory it is believed that long double strandedRNA introduced into cells is broken down into siRNA by a Type IIIendonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485).Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect theinvention relates to a single stranded RNA (siRNA) generated within acell and which promotes the formation of a RISC complex to effectsilencing of the target gene, i.e., a Serpinc1 gene. Accordingly, theterm “siRNA” is also used herein to refer to an RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded siRNAthat is introduced into a cell or organism to inhibit a target mRNA. Thesingle-stranded siRNAs are generally 15-30 nucleotides and arechemically modified. The design and testing of single-stranded siRNAsare described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell150: 883-894, the entire contents of each of which are herebyincorporated herein by reference. Any of the antisense nucleotidesequences described herein may be used as a single-stranded siRNA asdescribed herein or as chemically modified by the methods described inLima et al., (2012) Cell 150; 883-894.

In another aspect, the agent is a single-stranded antisense RNA moleculethat inhibits a target via an antisense inhibition mechanism. Thesingle-stranded antisense RNA molecule is complementary to a sequencewithin the target mRNA. Antisense RNA can inhibit translation in astoichiometric manner by base pairing to the mRNA and physicallyobstructing the translation machinery, see Dias, N. et al., (2002) MolCancer Ther 1:347-355. Alternatively, the single-stranded antisense RNAmolecule inhibits a target mRNA by hydridizing to the target andcleaving the target through an RNaseH cleavage event. Thesingle-stranded antisense RNA molecule may be about 15 to about 30nucleotides in length and have a sequence that is complementary to atarget sequence. For example, the single-stranded antisense RNA moleculemay comprise a sequence that is at least about 15, 16, 17, 18, 19, 20,or more contiguous nucleotides from any one of the antisense sequencesin any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21.

In another embodiment, an “iRNA” for use in the compositions, uses, andmethods of the invention is a double-stranded RNA and is referred toherein as a “double stranded RNAi agent,” “double-stranded RNA (dsRNA)molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary nucleicacid strands, referred to as having “sense” and “antisense” orientationswith respect to a target RNA, i.e., a Serpinc1 gene. In some embodimentsof the invention, a double-stranded RNA (dsRNA) triggers the degradationof a target RNA, e.g., an mRNA, through a post-transcriptionalgene-silencing mechanism referred to herein as RNA interference or RNAi.

The duplex region may be of any length that permits specific degradationof a desired target RNA through a RISC pathway, and may range from about9 to 36 base pairs in length, e.g., about 15-30 base pairs in length,for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 basepairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 basepairs in length. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Insome embodiments, the hairpin loop can comprise at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 20, at least 23 or more unpaired nucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not, but canbe covalently connected. Where the two strands are connected covalentlyby means other than an uninterrupted chain of nucleotides between the3′-end of one strand and the 5′-end of the respective other strandforming the duplex structure, the connecting structure is referred to asa “linker.” The RNA strands may have the same or a different number ofnucleotides. The maximum number of base pairs is the number ofnucleotides in the shortest strand of the dsRNA minus any overhangs thatare present in the duplex. In addition to the duplex structure, an RNAimay comprise one or more nucleotide overhangs.

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′-end, 3′-end orboth ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide,overhang at the 3′-end and/or the 5′-end. In one embodiment, the sensestrand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. Inanother embodiment, one or more of the nucleotides in the overhang isreplaced with a nucleoside thiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to adsRNA mean that there are no unpaired nucleotides or nucleotide analogsat a given terminal end of a dsRNA, i.e., no nucleotide overhang. One orboth ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt,the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNAis a dsRNA that is blunt at both ends, i.e., no nucleotide overhang ateither end of the molecule. Most often such a molecule will bedouble-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., a Serpinc1 mRNA. As usedherein, the term “region of complementarity” refers to the region on theantisense strand that is substantially complementary to a sequence, forexample a target sequence, e.g., a Serpinc1 nucleotide sequence, asdefined herein. Where the region of complementarity is not fullycomplementary to the target sequence, the mismatches can be in theinternal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 5, 4, 3,or 2 nucleotides of the 5′- and/or 3′-terminus of the iRNA.

The term “sense strand,” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g.,“Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) ColdSpring Harbor Laboratory Press). Other conditions, such asphysiologically relevant conditions as can be encountered inside anorganism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an iRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding Serpinc1). For example, apolynucleotide is complementary to at least a part of a Serpinc1 mRNA ifthe sequence is substantially complementary to a non-interrupted portionof an mRNA encoding Serpinc1.

In general, the majority of nucleotides of each strand areribonucleotides, but as described in detail herein, each or both strandscan also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, an “iRNA”may include ribonucleotides with chemical modifications. Suchmodifications may include all types of modifications disclosed herein orknown in the art. Any such modifications, as used in an iRNA molecule,are encompassed by “iRNA” for the purposes of this specification andclaims.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating,” “suppressing” and othersimilar terms, and includes any level of inhibition.

The phrase “inhibiting expression of a Serpinc1,” as used herein,includes inhibition of expression of any Serpinc1 gene (such as, e.g., amouse Serpinc1 gene, a rat Serpinc1 gene, a monkey Serpinc1 gene, or ahuman Serpinc1 gene) as well as variants or mutants of a Serpinc1 genethat encodes a Serpinc1 protein.

“Inhibiting expression of a Serpinc1 gene” includes any level ofinhibition of a Serpinc1 gene, e.g., at least partial suppression of theexpression of a Serpinc1 gene, such as an inhibition by at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, or at least about 99%.

The expression of a Serpinc1 gene may be assessed based on the level ofany variable associated with Serpinc1 gene expression, e.g., Serpinc1mRNA level, Serpinc1 protein level, or, for example,thrombin:antithrombin complex levels as a measure of thrombin generationportential, bleeding time, prothrombin time (PT), platelet count, and/oractivated partial thromboplastin time (aPTT). Inhibition may be assessedby a decrease in an absolute or relative level of one or more of thesevariables compared with a control level. The control level may be anytype of control level that is utilized in the art, e.g., a pre-dosebaseline level, or a level determined from a similar subject, cell, orsample that is untreated or treated with a control (such as, e.g.,buffer only control or inactive agent control).

In one embodiment, at least partial suppression of the expression of aSerpinc1 gene, is assessed by a reduction of the amount of Serpinc1 mRNAwhich can be isolated from or detected in a first cell or group of cellsin which a Serpinc1 gene is transcribed and which has or have beentreated such that the expression of a Serpinc1 gene is inhibited, ascompared to a second cell or group of cells substantially identical tothe first cell or group of cells but which has or have not been sotreated (control cells). The degree of inhibition may be expressed interms of

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, asused herein, includes contacting a cell by any possible means.Contacting a cell with an RNAi agent includes contacting a cell in vitrowith the iRNA or contacting a cell in vivo with the iRNA. The contactingmay be done directly or indirectly. Thus, for example, the RNAi agentmay be put into physical contact with the cell by the individualperforming the method, or alternatively, the RNAi agent may be put intoa situation that will permit or cause it to subsequently come intocontact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the RNAi agent. Contacting a cell in vivo may be done, forexample, by injecting the RNAi agent into or near the tissue where thecell is located, or by injecting the RNAi agent into another area, e.g.,the bloodstream or the subcutaneous space, such that the agent willsubsequently reach the tissue where the cell to be contacted is located.For example, the RNAi agent may contain and/or be coupled to a ligand,e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g.,the liver. Combinations of in vitro and in vivo methods of contactingare also possible. For example, a cell may also be contacted in vitrowith an RNAi agent and subsequently transplanted into a subject.

In one embodiment, contacting a cell with an iRNA includes “introducing”or “delivering the iRNA into the cell” by facilitating or effectinguptake or absorption into the cell. Absorption or uptake of an iRNA canoccur through unaided diffusive or active cellular processes, or byauxiliary agents or devices. Introducing an iRNA into a cell may be invitro and/or in vivo. For example, for in vivo introduction, iRNA can beinjected into a tissue site or administered systemically. In vivodelivery can also be done by a beta-glucan delivery system, such asthose described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S.Publication No. 2005/0281781, the entire contents of which are herebyincorporated herein by reference. In vitro introduction into a cellincludes methods known in the art such as electroporation andlipofection. Further approaches are described herein below and/or areknown in the art.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipidlayer encapsulating a pharmaceutically active molecule, such as anucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA istranscribed. LNPs are described in, for example, U.S. Pat. Nos.6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents ofwhich are hereby incorporated herein by reference.

The term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALPis a vesicle of lipids coating a reduced aqueous interior comprising anucleic acid such as an iRNA or a plasmid from which an iRNA istranscribed. SNALPs are described, e.g., in U.S. Patent ApplicationPublication Nos. 20060240093, 20070135372, and in InternationalApplication No. WO 2009082817, the entire contents of which are herebyincorporated herein by reference. Examples of “SNALP” formulations aredescribed below.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or agoose). In an embodiment, the subject is a human, such as a human beingtreated or assessed for a disease, disorder or condition that wouldbenefit from reduction in Serpinc1 expression; a human at risk for adisease, disorder or condition that would benefit from reduction inSerpinc1 expression; a human having a disease, disorder or conditionthat would benefit from reduction in Serpinc1 expression; and/or humanbeing treated for a disease, disorder or condition that would benefitfrom reduction in Serpinc1 expression as described herein. As usedherein, the terms “treating” or “treatment” refer to a beneficial ordesired result including, but not limited to, alleviation oramelioration of one or more symptoms, diminishing the extent ofbleeding, stabilized (i.e., not worsening) state of bleeding,amelioration or palliation of the bleeding, whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival in the absence of treatment.

By “lower” in the context of a disease marker or symptom is meant astatistically significant decrease in such level. The decrease can be,for example, at least 10%, at least 20%, at least 30%, at least 40% ormore, and is preferably down to a level accepted as within the range ofnormal for an individual without such disorder.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder or condition thereof, that would benefit from areduction in expression of a Sertpinc1 gene, refers to a reduction inthe likelihood that a subject will develop a symptom associated with asuch a disease, disorder, or condition, e.g., a symptom such as a bleed.The likelihood of developing a bleed is reduced, for example, when anindividual having one or more risk factors for a bleed either fails todevelop a bleed or develops a bleed with less severity relative to apopulation having the same risk factors and not receiving treatment asdescribed herein. The failure to develop a disease, disorder orcondition, or the reduction in the development of a symptom associatedwith such a disease, disorder or condition (e.g., by at least about 10%on a clinically accepted scale for that disease or disorder), or theexhibition of delayed symptoms delayed (e.g., by days, weeks, months oryears) is considered effective prevention.

As used herein, the term “bleeding disorder” is a disease or disorderthat results in poor blood clotting and/or excessive bleeding. Ableeding disorder may be an inherited disorder, such as a hemophilia orvon Willebrand's disease, or an acquired disorder, associated with, forexample, disseminated intravascular coagulation, pregnancy-associatedeclampsia, vitamin K deficiency, an autoimmune disorder, inflammatorybowel disease, ulcerative colitis, a dermatologic disorder (e.g.,psoriasis, pemphigus), a respiratory disease (e.g., asthma, chronicobstructive pulmonary disease), an allergic drug reaction, e.g., theresult of medications, such as aspirin, heparin, and warfarin, diabetes,acute hepatitis B infection, acute hepatitis C infection, a malignancyor solid tumor (e.g., prostate, lung, colon, pancreas, stomach, bileduct, head and neck, cervix, breast, melanoma, kidney, and/or ahematologic malignancy). In one embodiment, an inherited bleedingdisorder is a hemophilia, e.g., hemophilia A, B, or C. In oneembodiment, a subject having an inherited bleeding disorder, e.g., ahemophilia, has developed inhibitors, e.g., alloantibody inhibitors, toreplacement coagulation therapies and is referred to herein as an“inhibitor subject.” In one embodiment, the inhibitor subject hashemophilia A. In another embodiment, the inhibitor subject hashemophilia B. In yet another embodiment, the inhibitor subject hashemophilia C.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjecthaving a bleeding disorder and bleeding, is sufficient to effecttreatment of the disease (e.g., by diminishing, ameliorating ormaintaining the existing disease or one or more symptoms of disease).The “therapeutically effective amount” may vary depending on the RNAiagent, how the agent is administered, the disease and its severity andthe history, age, weight, family history, genetic makeup, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an iRNA that, when administered to a subjecthaving a bleeding disorder but not bleeding, e.g., a subject having ableeding disorder and scheduled for surgery, is sufficient to prevent orameliorate the disease or one or more symptoms of the disease.Ameliorating the disease includes slowing the course of the disease orreducing the severity of later-developing disease. The “prophylacticallyeffective amount” may vary depending on the iRNA, how the agent isadministered, the degree of risk of disease, and the history, age,weight, family history, genetic makeup, the types of preceding orconcomitant treatments, if any, and other individual characteristics ofthe patient to be treated.

A “therapeutically effective amount” or “prophylactically effectiveamount” also includes an amount of an RNAi agent that produces somedesired local or systemic effect at a reasonable benefit/risk ratioapplicable to any treatment. iRNA employed in the methods of the presentinvention may be administered in a sufficient amount to produce areasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human subjects and animal subjects without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the subject being treated. Some examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)lubricating agents, such as magnesium state, sodium lauryl sulfate andtalc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; (22) bulking agents, such as polypeptides and aminoacids (23) serum component, such as serum albumin, HDL and LDL; and (22)other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, cerebrospinalfluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In some embodiments, a “sample derived from a subject”refers to blood or plasma drawn from the subject.

II. iRNAs OF THE INVENTION

Described herein are iRNAs which inhibit the expression of a Serpinc1gene. In one embodiment, the iRNA agent includes double-strandedribonucleic acid (dsRNA) molecules for inhibiting the expression of aSerpinc1 gene in a cell, such as a cell within a subject, e.g., amammal, such as a human having a bleeding disorder, e.g., an inheritedbleeding disorder. The dsRNA includes an antisense strand having aregion of complementarity which is complementary to at least a part ofan mRNA formed in the expression of a Serpinc1 gene. The region ofcomplementarity is about 30 nucleotides or less in length (e.g., about30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides orless in length). Upon contact with a cell expressing the Serpinc1 gene,the iRNA inhibits the expression of the Serpinc1 gene (e.g., a human, aprimate, a non-primate, or a bird Sertpinc1 gene) by at least about 10%as assayed by, for example, a PCR or branched DNA (bDNA)-based method,or by a protein-based method, such as by immunofluorescence analysis,using, for example, Western Blotting or flowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of a Serpinc1gene. The other strand (the sense strand) includes a region that iscomplementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. As described elsewhere herein and as known in the art, thecomplementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs inlength, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23,15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27,18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28,19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29,20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29,21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length.Ranges and lengths intermediate to the above recited ranges and lengthsare also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence isbetween 15 and 30 nucleotides in length, e.g., between 15-29, 15-28,15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18,15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22,18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23,19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24,21-23, or 21-22 nucleotides in length. Ranges and lengths intermediateto the above recited ranges and lengths are also contemplated to be partof the invention.

In some embodiments, the dsRNA is between about 15 and about 20nucleotides in length, or between about 25 and about 30 nucleotides inlength. In general, the dsRNA is long enough to serve as a substrate forthe Dicer enzyme. For example, it is well-known in the art that dsRNAslonger than about 21-23 nucleotides in length may serve as substratesfor Dicer. As the ordinarily skilled person will also recognize, theregion of an RNA targeted for cleavage will most often be part of alarger RNA molecule, often an mRNA molecule. Where relevant, a “part” ofan mRNA target is a contiguous sequence of an mRNA target of sufficientlength to allow it to be a substrate for RNAi-directed cleavage (i.e.,cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of about 9to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36,9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34,12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33,15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31,11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26,15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30,18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20,19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21,19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22,20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22base pairs. Thus, in one embodiment, to the extent that it becomesprocessed to a functional duplex, of e.g., 15-30 base pairs, thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not anaturally occurring miRNA. In another embodiment, an iRNA agent usefulto target Serpinc1 expression is not generated in the target cell bycleavage of a larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides.dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties relative to their blunt-endedcounterparts. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′-end, 3′-end or both ends of either an antisense orsense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc.

iRNA compounds of the invention may be prepared using a two-stepprocedure. First, the individual strands of the double-stranded RNAmolecule are prepared separately. Then, the component strands areannealed. The individual strands of the siRNA compound can be preparedusing solution-phase or solid-phase organic synthesis or both. Organicsynthesis offers the advantage that the oligonucleotide strandscomprising unnatural or modified nucleotides can be easily prepared.Single-stranded oligonucleotides of the invention can be prepared usingsolution-phase or solid-phase organic synthesis or both.

In one aspect, a dsRNA of the invention includes at least two nucleotidesequences, a sense sequence and an anti-sense sequence. The sense strandis selected from the group of sequences provided in any one of Tables 3,4, 8, 11, 12, 14, 15, 20, and 21, and the corresponding antisense strandof the sense strand is selected from the group of sequences of any oneof Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21. In this aspect, one ofthe two sequences is complementary to the other of the two sequences,with one of the sequences being substantially complementary to asequence of an mRNA generated in the expression of a Serpinc1 gene. Assuch, in this aspect, a dsRNA will include two oligonucleotides, whereone oligonucleotide is described as the sense strand in any one ofTables 3, 4, 8, 11, 12, 14, 15, 20, and 21, and the secondoligonucleotide is described as the corresponding antisense strand ofthe sense strand in any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and21. In one embodiment, the substantially complementary sequences of thedsRNA are contained on separate oligonucleotides. In another embodiment,the substantially complementary sequences of the dsRNA are contained ona single oligonucleotide.

It will be understood that, although some of the sequences in Tables 3,4, 8, 11, 12, 14, 15, 20, and 21 are described as modified and/orconjugated sequences, the RNA of the iRNA of the invention e.g., a dsRNAof the invention, may comprise any one of the sequences set forth inTables 3, 4, 8, 11, 12, 14, 15, 20, and 21 that is un-modified,un-conjugated, and/or modified and/or conjugated differently thandescribed therein.

The skilled person is well aware that dsRNAs having a duplex structureof between about 20 and 23 base pairs, e.g., 21, base pairs have beenhailed as particularly effective in inducing RNA interference (Elbashiret al., EMBO 2001, 20:6877-6888). However, others have found thatshorter or longer RNA duplex structures can also be effective (Chu andRana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226).In the embodiments described above, by virtue of the nature of theoligonucleotide sequences provided in any one of Tables 3, 4, 8, 11, 12,14, 15, 20, and 21, dsRNAs described herein can include at least onestrand of a length of minimally 21 nucleotides. It can be reasonablyexpected that shorter duplexes having one of the sequences of any one ofTables 3, 4, 8, 11, 12, 14, 15, 20, and 21 minus only a few nucleotideson one or both ends can be similarly effective as compared to the dsRNAsdescribed above. Hence, dsRNAs having a sequence of at least 15, 16, 17,18, 19, 20, or more contiguous nucleotides derived from one of thesequences of any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, anddiffering in their ability to inhibit the expression of a Serpinc1 geneby not more than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNAcomprising the full sequence, are contemplated to be within the scope ofthe present invention.

In addition, the RNAs provided in any one of Tables 3, 4, 8, 11, 12, 14,15, 20, and 21 identify a site(s) in a Serpinc1 transcript that issusceptible to RISC-mediated cleavage. As such, the present inventionfurther features iRNAs that target within one of these sites. As usedherein, an iRNA is said to target within a particular site of an RNAtranscript if the iRNA promotes cleavage of the transcript anywherewithin that particular site. Such an iRNA will generally include atleast about 15 contiguous nucleotides from one of the sequences providedin any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21 coupled toadditional nucleotide sequences taken from the region contiguous to theselected sequence in a Serpinc1 gene.

While a target sequence is generally about 15-30 nucleotides in length,there is wide variation in the suitability of particular sequences inthis range for directing cleavage of any given target RNA. Varioussoftware packages and the guidelines set out herein provide guidance forthe identification of optimal target sequences for any given genetarget, but an empirical approach can also be taken in which a “window”or “mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that canserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aniRNA agent, mediate the best inhibition of target gene expression. Thus,while the sequences identified, for example, in any one of Tables 3, 4,8, 11, 12, 14, 15, 20, and 21 represent effective target sequences, itis contemplated that further optimization of inhibition efficiency canbe achieved by progressively “walking the window” one nucleotideupstream or downstream of the given sequences to identify sequences withequal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., inany one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, furtheroptimization could be achieved by systematically either adding orremoving nucleotides to generate longer or shorter sequences and testingthose sequences generated by walking a window of the longer or shortersize up or down the target RNA from that point. Again, coupling thisapproach to generating new candidate targets with testing foreffectiveness of iRNAs based on those target sequences in an inhibitionassay as known in the art and/or as described herein can lead to furtherimprovements in the efficiency of inhibition. Further still, suchoptimized sequences can be adjusted by, e.g., the introduction ofmodified nucleotides as described herein or as known in the art,addition or changes in overhang, or other modifications as known in theart and/or discussed herein to further optimize the molecule (e.g.,increasing serum stability or circulating half-life, increasing thermalstability, enhancing transmembrane delivery, targeting to a particularlocation or cell type, increasing interaction with silencing pathwayenzymes, increasing release from endosomes) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to thetarget sequence. In one embodiment, an iRNA as described herein containsno more than 3 mismatches. If the antisense strand of the iRNA containsmismatches to a target sequence, it is preferable that the area ofmismatch is not located in the center of the region of complementarity.If the antisense strand of the iRNA contains mismatches to the targetsequence, it is preferable that the mismatch be restricted to be withinthe last 5 nucleotides from either the 5′- or 3′-end of the region ofcomplementarity. For example, for a 23 nucleotide iRNA agent the strandwhich is complementary to a region of a Serpinc1 gene, generally doesnot contain any mismatch within the central 13 nucleotides. The methodsdescribed herein or methods known in the art can be used to determinewhether an iRNA containing a mismatch to a target sequence is effectivein inhibiting the expression of a Serpinc1 gene. Consideration of theefficacy of iRNAs with mismatches in inhibiting expression of a Serpinc1gene is important, especially if the particular region ofcomplementarity in a Serpinc1 gene is known to have polymorphic sequencevariation within the population.

III. MODIFIED iRNAs OF THE INVENTION

In one embodiment, the RNA of the iRNA of the invention e.g., a dsRNA,is un-modified, and does not comprise, e.g., chemical modificationsand/or conjugations known in the art and described herein. In anotherembodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, ischemically modified to enhance stability or other beneficialcharacteristics. The nucleic acids featured in the invention can besynthesized and/or modified by methods well established in the art, suchas those described in “Current protocols in nucleic acid chemistry,”Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y.,USA, which is hereby incorporated herein by reference. Modificationsinclude, for example, end modifications, e.g., 5′-end modifications(phosphorylation, conjugation, inverted linkages) or 3′-endmodifications (conjugation, DNA nucleotides, inverted linkages, etc.);base modifications, e.g., replacement with stabilizing bases,destabilizing bases, or bases that base pair with an expanded repertoireof partners, removal of bases (abasic nucleotides), or conjugated bases;sugar modifications (e.g., at the 2′-position or 4′-position) orreplacement of the sugar; and/or backbone modifications, includingmodification or replacement of the phosphodiester linkages. Specificexamples of iRNA compounds useful in the embodiments described hereininclude, but are not limited to RNAs containing modified backbones or nonatural internucleoside linkages. RNAs having modified backbonesinclude, among others, those that do not have a phosphorus atom in thebackbone. For the purposes of this specification, and as sometimesreferenced in the art, modified RNAs that do not have a phosphorus atomin their internucleoside backbone can also be considered to beoligonucleosides. In some embodiments, a modified iRNA will have aphosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use iniRNAs, in which both the sugar and the internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, an RNAmimetic that has been shown to have excellent hybridization properties,is referred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar backbone of an RNA is replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. Representative U.S. patents thatteach the preparation of PNA compounds include, but are not limited to,U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contentsof each of which are hereby incorporated herein by reference. AdditionalPNA compounds suitable for use in the iRNAs of the invention aredescribed in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known asa methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in Modified Nucleosides in Biochemistry, Biotechnology andMedicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Researchand Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRCPress, 1993. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the oligomeric compounds featured inthe invention. These include 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., dsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are exemplary base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

Representative U.S. patents that teach the preparation of locked nucleicacid nucleotides include, but are not limited to, the following: U.S.Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207;7,084,125; and 7,399,845, the entire contents of each of which arehereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

IV. iRNAs CONJUGATED TO LIGANDS

Another modification of the RNA of an iRNA of the invention involveschemically linking to the RNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the iRNA. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al.,Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g.,beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993,3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanovet al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie,1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995,14:969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or alipid. The ligand can also be a recombinant or synthetic molecule, suchas a synthetic polymer, e.g., a synthetic polyamino acid. Examples ofpolyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucoseamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a pharmacokinetic modulator (PK modulator). PK modulatorsinclude lipophiles, bile acids, steroids, phospholipid analogues,peptides, protein binding agents, PEG, vitamins etc. Exemplary PKmodulators include, but are not limited to, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotinetc. Oligonucleotides that comprise a number of phosphorothioatelinkages are also known to bind to serum protein, thus shortoligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15bases or 20 bases, comprising multiple of phosphorothioate linkages inthe backbone are also amenable to the present invention as ligands (e.g.as PK modulating ligands). In addition, aptamers that bind serumcomponents (e.g. serum proteins) are also suitable for use as PKmodulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesizedby the use of an oligonucleotide that bears a pendant reactivefunctionality, such as that derived from the attachment of a linkingmolecule onto the oligonucleotide (described below). This reactiveoligonucleotide may be reacted directly with commercially-availableligands, ligands that are synthesized bearing any of a variety ofprotecting groups, or ligands that have a linking moiety attachedthereto.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the oligonucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, naproxen oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as liver cells. Also included are HSA and low densitylipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 9). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 10) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glycosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, a α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, aniRNA oligonucleotide further comprises a carbohydrate. The carbohydrateconjugated iRNA are advantageous for the in vivo delivery of nucleicacids, as well as compositions suitable for in vivo therapeutic use, asdescribed herein. As used herein, “carbohydrate” refers to a compoundwhich is either a carbohydrate per se made up of one or moremonosaccharide units having at least 6 carbon atoms (which can belinear, branched or cyclic) with an oxygen, nitrogen or sulfur atombonded to each carbon atom; or a compound having as a part thereof acarbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C5and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., C5, C6,C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is a monosaccharide. In one embodiment, themonosaccharide is an N-acetylgalactosamine, such as

In another embodiment, a carbohydrate conjugate for use in thecompositions and methods of the invention is selected from the groupconsisting of:

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator and/or a cell permeation peptide.

D. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an iRNA oligonucleotide with various linkers that can becleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular iRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)—O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—,—S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—.These candidates can be evaluated using methods analogous to thosedescribed above.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O), where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In one embodiment, an iRNA of the invention is conjugated to acarbohydrate through a linker. Non-limiting examples of iRNAcarbohydrate conjugates with linkers of the compositions and methods ofthe invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalentor trivalent branched linker selected from the group of structures shownin any of formula (XXXI)-(XXXIV):

wherein:q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different;P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherin one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), CC or C(O);R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H oramino acid side chain. Trivalent conjugating GalNAc derivatives areparticularly useful for use with RNAi agents for inhibiting theexpression of a target gene, such as those of formula (XXXV):

-   -   wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide,        such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of thisinvention, are iRNA compounds, preferably dsRNAs, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAstypically contain at least one region wherein the RNA is modified so asto confer upon the iRNA increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the iRNA can serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof iRNA inhibition of gene expression. Consequently, comparable resultscan often be obtained with shorter iRNAs when chimeric dsRNAs are used,compared to phosphorothioate deoxy dsRNAs hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction can be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

IV. DELIVERY OF AN iRNA OF THE INVENTION

The delivery of an iRNA of the invention to a cell e.g., a cell within asubject, such as a human subject (e.g., a subject in need thereof, suchas a subject having a bleeding disorder) can be achieved in a number ofdifferent ways. For example, delivery may be performed by contacting acell with an iRNA of the invention either in vitro or in vivo. In vivodelivery may also be performed directly by administering a compositioncomprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivodelivery may be performed indirectly by administering one or morevectors that encode and direct the expression of the iRNA. Thesealternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an iRNA of the invention (seee.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144and WO94/02595, which are incorporated herein by reference in theirentireties). For in vivo delivery, factors to consider in order todeliver an iRNA molecule include, for example, biological stability ofthe delivered molecule, prevention of non-specific effects, andaccumulation of the delivered molecule in the target tissue. Thenon-specific effects of an iRNA can be minimized by localadministration, for example, by direct injection or implantation into atissue or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J., et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA compositionto the target tissue and avoid undesirable off-target effects. iRNAmolecules can be modified by chemical conjugation to lipophilic groupssuch as cholesterol to enhance cellular uptake and prevent degradation.For example, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., etal (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer hasbeen shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O., et al (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H., et al (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328;Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., etal (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA formsa complex with cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the Serpinc1 gene can be expressed from transcriptionunits inserted into DNA or RNA vectors (see, e.g., Couture, A, et al.,TIG. (1996), 12:5-10; Skillern, A., et al., International PCTPublication No. WO 00/22113, Conrad, International PCT Publication No.WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can betransient (on the order of hours to weeks) or sustained (weeks to monthsor longer), depending upon the specific construct used and the targettissue or cell type. These transgenes can be introduced as a linearconstruct, a circular plasmid, or a viral vector, which can be anintegrating or non-integrating vector. The transgene can also beconstructed to permit it to be inherited as an extrachromosomal plasmid(Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as acomplex with cationic lipid carriers (e.g., Oligofectamine) ornon-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipidtransfections for iRNA-mediated knockdowns targeting different regionsof a target RNA over a period of a week or more are also contemplated bythe invention. Successful introduction of vectors into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofcells ex vivo can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are further describedbelow.

Vectors useful for the delivery of an iRNA will include regulatoryelements (promoter, enhancer, etc.) sufficient for expression of theiRNA in the desired target cell or tissue. The regulatory elements canbe chosen to provide either constitutive or regulated/inducibleexpression.

Expression of the iRNA can be precisely regulated, for example, by usingan inducible regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of dsRNA expression in cells or inmammals include, for example, regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the iRNA transgene.

Viral vectors that contain nucleic acid sequences encoding an iRNA canbe used. For example, a retroviral vector can be used (see Miller etal., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectorscontain the components necessary for the correct packaging of the viralgenome and integration into the host cell DNA. The nucleic acidsequences encoding an iRNA are cloned into one or more vectors, whichfacilitate delivery of the nucleic acid into a patient. More detailabout retroviral vectors can be found, for example, in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993). Lentiviral vectors contemplated for use include, forexample, the HIV based vectors described in U.S. Pat. Nos. 6,143,520;5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs of theinvention. Adenoviruses are especially attractive vehicles, e.g., fordelivering genes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitableAV vector for expressing an iRNA featured in the invention, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Adeno-associated virus (AAV) vectors may also be used to delivery aniRNA of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med.204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, theiRNA can be expressed as two separate, complementary single-stranded RNAmolecules from a recombinant AAV vector having, for example, either theU6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. SuitableAAV vectors for expressing the dsRNA featured in the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent ApplicationNo. WO 94/13788; and International Patent Application No. WO 93/24641,the entire disclosures of which are herein incorporated by reference.

Another viral vector suitable for delivery of an iRNA of the inventionis a pox virus such as a vaccinia virus, for example an attenuatedvaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such asfowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectorswith envelope proteins or other surface antigens from other viruses, orby substituting different viral capsid proteins, as appropriate. Forexample, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes; see, e.g.,Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosureof which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

V. PHARMACEUTICAL COMPOSITIONS OF THE INVENTION

The present invention also includes pharmaceutical compositions andformulations which include the iRNAs of the invention. In oneembodiment, provided herein are pharmaceutical compositions containingan iRNA, as described herein, and a pharmaceutically acceptable carrier.The pharmaceutical compositions containing the iRNA are useful fortreating a disease or disorder associated with the expression oractivity of a Serpinc1 gene, e.g. a bleeding disorder.

Such pharmaceutical compositions are formulated based on the mode ofdelivery. One example is compositions that are formulated for systemicadministration via parenteral delivery, e.g., by intravenous (IV)delivery. Another example is compositions that are formulated for directdelivery into the brain parenchyma, e.g., by infusion into the brain,such as by continuous pump infusion. The pharmaceutical compositions ofthe invention may be administered in dosages sufficient to inhibitexpression of a Serpinc1 gene. In general, a suitable dose of an iRNA ofthe invention will be in the range of about 0.001 to about 200.0milligrams per kilogram body weight of the recipient per day, generallyin the range of about 1 to 50 mg per kilogram body weight per day. Forexample, the dsRNA can be administered at about 0.01 mg/kg, about 0.05mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg,about 3 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40mg/kg, or about 50 mg/kg per single dose.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In another embodiment, the dsRNA is administered at a dose of about 0.1to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

For example, the dsRNA may be administered at a dose of about 0.01,0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

In another embodiment, the dsRNA is administered at a dose of about 0.5to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20mg/kg, or about 15 to about 20 mg/kg. In one embodiment, the dsRNA isadministered at a dose of about 10 mg/kg to about 30 mg/kg. Values andranges intermediate to the recited values are also intended to be partof this invention.

For example, subjects can be administered a therapeutic amount of iRNA,such as about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5,13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5,20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5,27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention.

The pharmaceutical composition can be administered once daily, or theiRNA can be administered as two, three, or more sub-doses at appropriateintervals throughout the day or even using continuous infusion ordelivery through a controlled release formulation. In that case, theiRNA contained in each sub-dose must be correspondingly smaller in orderto achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of theiRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that subsequent doses are administered at notmore than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4week intervals. In some embodiments of the invention, a single dose ofthe pharmaceutical compositions of the invention is administered onceper week. In other embodiments of the invention, a single dose of thepharmaceutical compositions of the invention is administered bi-monthly.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as a bleeding disorder thatwould benefit from reduction in the expression of Serpinc1. Such modelscan be used for in vivo testing of iRNA, as well as for determining atherapeutically effective dose. Suitable mouse models are known in theart and include, for example, Hemophilia A mouse models and HemohphiliaB mouse models, e.g., mice containing a knock-out of a clotting factorgene, such as those described in Bolliger, et al. (2010) Thromb Haemost103:1233-1238, Bi L, et al. (1995) Nat Genet 10: 119-21, Lin et al.(1997) Blood 90: 3962-6, Kundu et al. (1998) Blood 92: 168-74, Wang etal. (1997) Proc Natl Acad Sci USA 94: 11563-6, and Jin, et al. (2004)Blood 104:1733.

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration The iRNA can be delivered in a manner to target aparticular tissue, such as the liver (e.g., the hepatocytes of theliver). Pharmaceutical compositions and formulations for topicaladministration can include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like can be necessary or desirable. Coated condoms,gloves and the like can also be useful. Suitable topical formulationsinclude those in which the iRNAs featured in the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Suitable lipids and liposomes include neutral (e.g.,dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.,dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.,dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). iRNAs featured in the invention can be encapsulatedwithin liposomes or can form complexes thereto, in particular tocationic liposomes. Alternatively, iRNAs can be complexed to lipids, inparticular to cationic lipids. Suitable fatty acids and esters includebut are not limited to arachidonic acid, oleic acid, eicosanoic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof). Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can beformulated for delivery in a membranous molecular assembly, e.g., aliposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the iRNA composition. The lipophilic material isolates theaqueous interior from an aqueous exterior, which typically does notinclude the iRNA composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the iRNA are delivered into the cell where theiRNA can specifically bind to a target RNA and can mediate RNAi. In somecases the liposomes are also specifically targeted, e.g., to direct theiRNA to particular cell types.

A liposome containing a RNAi agent can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAiagent preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the RNAi agentand condense around the RNAi agent to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of RNAi agent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad.Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No.5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al.Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci.75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984;Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Thesemethods are readily adapted to packaging RNAi agent preparations intoliposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleicacids rather than complex with it. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH-sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al., Journal of Controlled Release, 1992, 19,269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550,1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human GeneTher. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J.11:417, 1992.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver RNAi agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated RNAi agents in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of RNAi agent (see, e.g.,Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 andU.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer RNAi agent into the skin. In some implementations,liposomes are used for delivering RNAi agent to epidermal cells and alsoto enhance the penetration of RNAi agent into dermal tissues, e.g., intoskin. For example, the liposomes can be applied topically. Topicaldelivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992,vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992,259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690,1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth.Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth.Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad.Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith RNAi agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include RNAi agentcan be delivered, for example, subcutaneously by infection in order todeliver RNAi agent to keratinocytes in the skin. In order to crossintact mammalian skin, lipid vesicles must pass through a series of finepores, each with a diameter less than 50 nm, under the influence of asuitable transdermal gradient. In addition, due to the lipid properties,these transferosomes can be self-optimizing (adaptive to the shape ofpores, e.g., in the skin), self-repairing, and can frequently reachtheir targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. Nos. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided asmicellar formulations. “Micelles” are defined herein as a particulartype of molecular assembly in which amphipathic molecules are arrangedin a spherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the siRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe siRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the siRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in alipid formulation, e.g., a LNP, e.g., to form a SPLP, pSPLP, SNALP, orother nucleic acid-lipid particle.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle, including SPLP. As used herein, the term “SPLP” refers to anucleic acid-lipid particle comprising plasmid DNA encapsulated within alipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, anon-cationic lipid, and a lipid that prevents aggregation of theparticle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremelyuseful for systemic applications, as they exhibit extended circulationlifetimes following intravenous (i.v.) injection and accumulate atdistal sites (e.g., sites physically separated from the administrationsite). SPLPs include “pSPLP,” which include an encapsulated condensingagent-nucleic acid complex as set forth in PCT Publication No. WO00/03683. The particles of the present invention typically have a meandiameter of about 50 nm to about 150 nm, more typically about 60 nm toabout 130 nm, more typically about 70 nm to about 110 nm, most typicallyabout 70 nm to about 90 nm, and are substantially nontoxic. In addition,the nucleic acids when present in the nucleic acid-lipid particles ofthe present invention are resistant in aqueous solution to degradationwith a nuclease. Nucleic acid-lipid particles and their method ofpreparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501;6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010/0324120 andPCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. Ranges intermediate to the above recited ranges are alsocontemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which isincorporated herein by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml;Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are described in Table 1.

TABLE 1 cationic lipid/non-cationic lipid/cholesterol/PEG-lipidconjugate Ionizable/Cationic Lipid Lipid:siRNA ratio SNALP-11,2-Dilinolenyloxy-N,N-dimethylaminopropaneDLinDMA/DPPC/Cholesterol/PEG-cDMA (DLinDMA) (57.1/7.1/34.4/1.4)lipid:siRNA~7:1 2-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DPPC/Cholesterol/PEG-cDMA dioxolane (XTC) 57.1/7.1/34.4/1.4lipid:siRNA~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5lipid:siRNA~6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5lipid:siRNA~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5,lipid:siRNA~6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5,lipid:siRNA~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-ALN100/DSPC/Cholesterol/PEG-DMG octadeca-9,12-dienyl)tetrahydro-3aH-50/10/38.5/1.5 cyclopenta[d][1,3]dioxol-5-amine (ALN100) Lipid:siRNA10:1 LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-MC-3/DSPC/Cholesterol/PEG-DMG tetraen-19-yl 4-(dimethylamino)butanoate50/10/38.5/1.5 (MC3) Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2-Tech G1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:siRNA 10:1yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC:distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18)(PEG with avg mol wt of 2000) PEG-cDMA:PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No.61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun.10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009;U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, andInternational Application No. PCT/US2010/022614, filed Jan. 29, 2010,which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. Publication No.2010/0324120, filed Jun. 10, 2010, the entire contents of which arehereby incorporated by reference.

ALNY-100 comprising formulations are described, e.g., Internationalpatent application number PCT/US09/63933, filed on Nov. 10, 2009, whichis hereby incorporated by reference.

C12-200 comprising formulations are described in U.S. Provisional Ser.No. 61/175,770, filed May 5, 2009 and International Application No.PCT/US10/33777, filed May 5, 2010, which are hereby incorporated byreference.

Synthesis of Ionizable/Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in thenucleic acid-lipid particles of the invention can be prepared by knownorganic synthesis techniques, including the methods described in moredetail in the Examples. All substituents are as defined below unlessindicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms can be optionally oxidized, and the nitrogenheteroatom can be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle can be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy,—SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same ordifferent and independently hydrogen, alkyl or heterocycle, and each ofsaid alkyl and heterocycle substituents can be further substituted withone or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy,—NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and—SOnNRxRy.

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods of the invention can require the use ofprotecting groups. Protecting group methodology is well known to thoseskilled in the art (see, for example, Protective Groups in OrganicSynthesis, Green, T. W. et al., Wiley-Interscience, New York City,1999). Briefly, protecting groups within the context of this inventionare any group that reduces or eliminates unwanted reactivity of afunctional group. A protecting group can be added to a functional groupto mask its reactivity during certain reactions and then removed toreveal the original functional group. In some embodiments an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

Synthesis of Formula A

In some embodiments, nucleic acid-lipid particles of the invention areformulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can beoptionally substituted, and R3 and R4 are independently lower alkyl orR3 and R4 can be taken together to form an optionally substitutedheterocyclic ring. In some embodiments, the cationic lipid is XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, thelipid of formula A above can be made by the following Reaction Schemes 1or 2, wherein all substituents are as defined above unless indicatedotherwise.

Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl,each can be optionally substituted, and R3 and R4 are independentlylower alkyl or R3 and R4 can be taken together to form an optionallysubstituted heterocyclic ring, can be prepared according to Scheme 1.Ketone 1 and bromide 2 can be purchased or prepared according to methodsknown to those of ordinary skill in the art. Reaction of 1 and 2 yieldsketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.The lipids of formula A can be converted to the corresponding ammoniumsalt with an organic salt of formula 5, where X is anion counter ionselected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared accordingto Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased orprepared according to methods known to those of ordinary skill in theart. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to thecorresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) was as follows. A solution of(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),4-N,N-dimethylaminopyridine (0.61 g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) indichloromethane (5 mL) was stirred at room temperature overnight. Thesolution was washed with dilute hydrochloric acid followed by diluteaqueous sodium bicarbonate. The organic fractions were dried overanhydrous magnesium sulphate, filtered and the solvent removed on arotovap. The residue was passed down a silica gel column (20 g) using a1-5% methanol/dichloromethane elution gradient. Fractions containing thepurified product were combined and the solvent removed, yielding acolorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the followingscheme 3:

Synthesis of 515

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 mlanhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogenatmosphere. After complete addition, reaction mixture was warmed to roomtemperature and then heated to reflux for 4 h. Progress of the reactionwas monitored by TLC. After completion of reaction (by TLC) the mixturewas cooled to 0° C. and quenched with careful addition of saturatedNa2SO4 solution. Reaction mixture was stirred for 4 h at roomtemperature and filtered off. Residue was washed well with THF. Thefiltrate and washings were mixed and diluted with 400 mL dioxane and 26mL conc. HCl and stirred for 20 minutes at room temperature. Thevolatilities were stripped off under vacuum to furnish the hydrochloridesalt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz):δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H),2.50-2.45 (m, 5H).

Synthesis of 516

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL twoneck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dryDCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO3solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4and the solvent was evaporated to give crude material which was purifiedby silica gel column chromatography to get 516 as sticky mass. Yield: 11g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H),5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m,2H). LC-MS [M+H] −232.3 (96.94%).

Synthesis of 517A and 517E

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of220 mL acetone and water (10:1) in a single neck 500 mL RBF and to itwas added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanolat room temperature. After completion of the reaction (˜3 h), themixture was quenched with addition of solid Na2SO3 and resulting mixturewas stirred for 1.5 h at room temperature. Reaction mixture was dilutedwith DCM (300 mL) and washed with water (2×100 mL) followed by saturatedNaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50mL). Organic phase was dried over an.Na2SO4 and solvent was removed invacuum. Silica gel column chromatographic purification of the crudematerial was afforded a mixture of diastereomers, which were separatedby prep HPLC. Yield: −6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz):δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H),3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS—[M+H]−266.3,[M+NH4+]−283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil.1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H),5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H),1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519

A solution of compound 518 (1 eq) in hexane (15 mL) was added in adrop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq).After complete addition, the mixture was heated at 40° C. over 0.5 hthen cooled again on an ice bath. The mixture was carefully hydrolyzedwith saturated aqueous Na2SO4 then filtered through celite and reducedto an oil. Column chromatography provided the pure 519 (1.3 g, 68%)which was obtained as a colorless oil. 13C NMR δ=130.2, 130.1 (×2),127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7,29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1;Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc.654.6. Found 654.6.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totaldsRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated dsRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” dsRNA content (as measured by thesignal in the absence of surfactant) from the total dsRNA content.Percent entrapped dsRNA is typically >85%. For SNALP formulation, theparticle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancersurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention can be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion canbe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention can be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

an RNAi agent of the invention may be incorporated into a particle,e.g., a microparticle. Microparticles can be produced by spray-drying,but may also be produced by other methods including lyophilization,evaporation, fluid bed drying, vacuum drying, or a combination of thesetechniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of iRNAs through themucosa is enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (seee.g., Malmsten, M. Surfactants and polymers in drug delivery, InformaHealth Care, New York, N.Y., 2002; Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption of iRNAsthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Suitable chelating agents include but are not limited todisodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(see e.g., Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of iRNAs through the alimentary mucosa (see e.g.,Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33). This class of penetration enhancers includes, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and non-steroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293fectin™(Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad,Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a)D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA compounds and (b) one or moreagents which function by a non-RNAi mechanism and which are useful intreating a bleeding disorder. Examples of such agents include, but arenot limited to an anti-inflammatory agent, anti-steatosis agent,anti-viral, and/or anti-fibrosis agent. In addition, other substancescommonly used to protect the liver, such as silymarin, can also be usedin conjunction with the iRNAs described herein. Other agents useful fortreating liver diseases include telbivudine, entecavir, and proteaseinhibitors such as telaprevir and other disclosed, for example, in Tunget al., U.S. Application Publication Nos. 2005/0148548, 2004/0167116,and 2003/0144217; and in Hale et al., U.S. Application Publication No.2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED50 with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby Serpinc1 expression. In any event, the administering physician canadjust the amount and timing of iRNA administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

VI. METHODS OF THE INVENTION

The present invention also provides methods of using an iRNA of theinvention and/or a composition containing an iRNA of the invention toreduce and/or inhibit Serpinc1 expression in a cell. In other aspects,the present invention provides an iRNA of the invention and/or acomposition comprising an iRNA of the invention for use in reducingand/or inhibiting Serpinc1 expression in a cell. In yet other aspects,use of an iRNA of the invention and/or a composition comprising an iRNAof the invention for the manufacture of a medicament for reducing and/orinhibiting Serpinc1 expression in a cell are provided.

The methods and uses include contacting the cell with an iRNA, e.g., adsRNA, of the invention and maintaining the cell for a time sufficientto obtain degradation of the mRNA transcript of a Serpinc1 gene, therebyinhibiting expression of the Serpinc1 gene in the cell.

Reduction in gene expression can be assessed by any methods known in theart. For example, a reduction in the expression of Serpinc1 may bedetermined by determining the mRNA expression level of Serpinc1 usingmethods routine to one of ordinary skill in the art, e.g., Northernblotting, qRT-PCR, by determining the protein level of Serpinc1 usingmethods routine to one of ordinary skill in the art, such as Westernblotting, immunological techniques, and/or by determining a biologicalactivity of Serpinc1, such as affecting one or more molecules associatedwith the cellular blood clotting mechanism (or in an in vivo setting,blood clotting itself).

In the methods and uses of the invention the cell may be contacted invitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may beany cell that expresses a Serpinc1 gene. A cell suitable for use in themethods and uses of the invention may be a mammalian cell, e.g., aprimate cell (such as a human cell or a non-human primate cell, e.g., amonkey cell or a chimpanzee cell), a non-primate cell (such as a cowcell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell,a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, adog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bearcell, or a buffalo cell), a bird cell (e.g., a duck cell or a goosecell), or a whale cell. In one embodiment, the cell is a human cell,e.g., a human liver cell.

Serpinc1 expression may be inhibited in the cell by at least about 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or about 100%.

The in vivo methods and uses of the invention may include administeringto a subject a composition containing an iRNA, where the iRNA includes anucleotide sequence that is complementary to at least a part of an RNAtranscript of the Serpinc1 gene of the mammal to be treated. When theorganism to be treated is a mammal such as a human, the composition canbe administered by any means known in the art including, but not limitedto oral, intraperitoneal, or parenteral routes, including intracranial(e.g., intraventricular, intraparenchymal and intrathecal), intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Incertain embodiments, the compositions are administered by intravenousinfusion or injection.

In some embodiments, the administration is via a depot injection. Adepot injection may release the iRNA in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof Serpinc1, or a therapeutic or prophylactic effect. A depot injectionmay also provide more consistent serum concentrations. Depot injectionsmay include subcutaneous injections or intramuscular injections. Inpreferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local orsystemic treatment is desired and based upon the area to be treated. Theroute and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods forinhibiting the expression of a Serpinc1 gene in a mammal, e.g., a human.The present invention also provides a composition comprising an iRNA,e.g., a dsRNA, that targets a Serpinc1 gene in a cell of a mammal foruse in inhibiting expression of the Serpinc1 gene in the mammal. Inanother aspect, the present invention provides use of an iRNA, e.g., adsRNA, that targets a Serpinc1 gene in a cell of a mammal in themanufacture of a medicament for inhibiting expression of the Serpinc1gene in the mammal.

The methods and uses include administering to the mammal, e.g., a human,a composition comprising an iRNA, e.g., a dsRNA, that targets a Serpinc1gene in a cell of the mammal and maintaining the mammal for a timesufficient to obtain degradation of the mRNA transcript of the Serpinc1gene, thereby inhibiting expression of the Serpinc1 gene in the mammal.

Reduction in gene expression can be assessed by any methods known it theart and by methods, e.g. qRT-PCR, described herein. Reduction in proteinproduction can be assessed by any methods known it the art and bymethods, e.g. ELISA, described herein. In one embodiment, a punctureliver biopsy sample serves as the tissue material for monitoring thereduction in Serpinc1 gene and/or protein expression. In anotherembodiment, a blood sample serves as the tissue material for monitoringthe reduction in Serpinc1 gene and/or protein expression. In otherembodiments, inhibition of the expression of a Serpinc1 gene ismonitored indirectly by, for example, determining the expression and/oractivity of a gene in a Serpinc1 pathway (see, e.g., FIG. 1). Forexample, the activity of factor Xa may be monitored to determine theinhibition of expression of a Serpinc1 gene. Antithrombin levels, clotformation, and/or endogenous thrombin potential, in a sample, e.g., ablood or liver sample, may also be measured. Suitable assays are furtherdescribed in the Examples section below.

The present invention further provides methods of treating a subjecthaving a disorder that would benefit from reduction in Serpinc1expression, e.g., hemophilia. The treatment methods (and uses) of theinvention include administering to the subject, e.g., a human, atherapeutically effective amount of an iRNA targeting a Serpinc1 gene ora pharmaceutical composition comprising an iRNA targeting a Serpinc1gene, thereby treating the subject having a disorder that would benefitfrom reduction in Serpinc1 expression.

In one aspect, the invention provides methods of preventing at least onesymptom in a subject having a disorder that would benefit from reductionin Serpinc1 expression. The methods include administering to the subjecta therapeutically effective amount of the iRNA, e.g., dsRNA, or vectorof the invention, thereby preventing at least one symptom in the subjecthaving a disorder that would benefit from reduction in Serpinc1expression. For example, the invention provides methods for preventingbleeding in a subject suffering from a disorder that would benefit fromreduction in Serpinc1 expression, e.g., a hemophilia

In another aspect, the present invention provides use of atherapeutically effective amount of an iRNA of the invention fortreating a subject, e.g., a subject that would benefit from a reductionand/or inhibition of Serpinc1 expression. The iRNA includes iRNAtargeting a Serpinc1 gene or a pharmaceutical composition comprising aniRNA targeting a Serpinc1 gene.

In yet another aspect, the present invention provides use of an iRNA ofthe invention targeting a Serpinc1 gene or a pharmaceutical compositioncomprising an iRNA targeting a Serpinc1 gene in the manufacture of amedicament for treating a subject, e.g., a subject that would benefitfrom a reduction and/or inhibition of Serpinc1 expression.

In another aspect, the invention provides uses of an iRNA, e.g., adsRNA, of the invention for preventing at least one symptom in a subjectsuffering from a disorder that would benefit from a reduction and/orinhibition of Serpinc1 expression, such as a bleeding disorder, e.g., ahemophilia.

In a further aspect, the present invention provides uses of an iRNA ofthe invention in the manufacture of a medicament for preventing at leastone symptom in a subject suffering from a disorder that would benefitfrom a reduction and/or inhibition of Serpinc1 expression, such as ableeding disorder, e.g., a hemophilia. An iRNA of the invention may beadministered in “naked” form, or as a “free iRNA.” A naked iRNA isadministered in the absence of a pharmaceutical composition. The nakediRNA may be in a suitable buffer solution. The buffer solution maycomprise acetate, citrate, prolamine, carbonate, or phosphate, or anycombination thereof. In one embodiment, the buffer solution is phosphatebuffered saline (PBS). The pH and osmolarity of the buffer solutioncontaining the iRNA can be adjusted such that it is suitable foradministering to a subject.

Alternatively, an iRNA of the invention may be administered as apharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition ofSerpinc1 gene expression are those having a bleeding disorder, e.g., aninherited bleeding disorder or an acquired bleeding disorder asdescribed herein. In one embodiment, a subject having an inheritedbleeding disorder has a hemophilia, e.g., hemophilia A, B, or C. In oneembodiment, a subject having an inherited bleeding disorder, e.g., ahemophilia, is an inhibitor subject. In one embodiment, the inhibitorsubject has hemophilia A. In another embodiment, the inhibitor subjecthas hemophilia B. In yet another embodiment, the inhibitor subject hashemophilia C. Treatment of a subject that would benefit from a reductionand/or inhibition of Serpinc1 gene expression includes therapeutic(e.g., on-demand, e.g., the subject is bleeding (spontaneous bleeding orbleeding as a result of trauma) and failing to clot) and prophylactic(e.g., the subject is not bleeding and/or is to undergo surgery)treatment.

The invention further provides methods and uses for the use of an iRNAor a pharmaceutical composition thereof, e.g., for treating a subjectthat would benefit from reduction and/or inhibition of Serpinc1expression, e.g., a subject having a bleeding disorder, in combinationwith other pharmaceuticals and/or other therapeutic methods, e.g., withknown pharmaceuticals and/or known therapeutic methods, such as, forexample, those which are currently employed for treating thesedisorders. For example, in certain embodiments, an iRNA targetingSerpinc1 is administered in combination with, e.g., an agent useful intreating a bleeding disorder as described elsewhere herein. For example,additional therapeutics and therapeutic methods suitable for treating asubject that would benefit from reduction in Serpinc1 expression, e.g.,a subject having a bleeding disorder, include fresh-frozen plasma (FFP);recombinant FVIIa; recombinant FIX; FXI concentrates; virus-inactivated,vWF-containing FVIII concentrates; desensitization therapy which mayinclude large doses of FVIII or FIX, along with steroids or intravenousimmunoglobulin (IVIG) and cyclophosphamide; plasmapheresis inconjunction with immunosuppression and infusion of FVIII or FIX, with orwithout antifibrinolytic therapy; immune tolerance induction (ITI), withor without immunosuppressive therapy (e.g., cyclophosphamide,prednisone, and/or anti-CD20); desmopressin acetate [DDAVP];antifibrinolytics, such as aminocaproic acid and tranexamic acid;activated prothrombin complex concentrate (PCC); antihemophilic agents;corticosteroids; immunosuppressive agents; and estrogens. The iRNA andan additional therapeutic agent and/or treatment may be administered atthe same time and/or in the same combination, e.g., parenterally, or theadditional therapeutic agent can be administered as part of a separatecomposition or at separate times and/or by another method known in theart or described herein.

In one embodiment, the methods and uses include administering acomposition featured herein such that expression of the target Serpinc1gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18,24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80hours. In one embodiment, expression of the target Serpinc1 gene isdecreased for an extended duration, e.g., at least about two, three,four, five, six, seven days or more, e.g., about one week, two weeks,three weeks, or about four weeks or longer.

Preferably, the iRNAs useful for the methods, uses, and compositionsfeatured herein specifically target RNAs (primary or processed) of thetarget Serpinc1 gene. Compositions, uses, and methods for inhibiting theexpression of these genes using iRNAs can be prepared and performed asdescribed herein.

Administration of the dsRNA according to the methods and uses of theinvention may result in a reduction of the severity, signs, symptoms,and/or markers of such diseases or disorders in a patient with ableeding disorder. By “reduction” in this context is meant astatistically significant decrease in such level. The reduction can be,for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, frequency of bleeds, reduction in pain, quality of life, doseof a medication required to sustain a treatment effect, level of adisease marker or any other measurable parameter appropriate for a givendisease being treated or targeted for prevention. It is well within theability of one skilled in the art to monitor efficacy of treatment orprevention by measuring any one of such parameters, or any combinationof parameters. For example, efficacy of treatment of a bleeding disordermay be assessed, for example, by periodic monitoring ofthrombin:anti-thrombin levels. Comparisons of the later readings withthe initial readings provide a physician an indication of whether thetreatment is effective. It is well within the ability of one skilled inthe art to monitor efficacy of treatment or prevention by measuring anyone of such parameters, or any combination of parameters. In connectionwith the administration of an iRNA targeting Serpinc1 or pharmaceuticalcomposition thereof, “effective against” a bleeding disorder indicatesthat administration in a clinically appropriate manner results in abeneficial effect for at least a statistically significant fraction ofpatients, such as a improvement of symptoms, a cure, a reduction indisease, extension of life, improvement in quality of life, or othereffect generally recognized as positive by medical doctors familiar withtreating bleeding disorders and the related causes.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given iRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in theseverity of disease as determined by one skilled in the art of diagnosisbased on a clinically accepted disease severity grading scale, as butone example the Child-Pugh score (sometimes the Child-Turcotte-Pughscore). Any positive change resulting in e.g., lessening of severity ofdisease measured using the appropriate scale, represents adequatetreatment using an iRNA or iRNA formulation as described herein.

Subjects can be administered a therapeutic amount of iRNA, such as about0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.1 mg/kg,0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg,0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6 mg/kg, 0.65 mg/kg, 0.7 mg/kg,0.75 mg/kg, 0.8 mg/kg, 0.85 mg/kg, 0.9 mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.1mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg,1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4mg/kg, 2.5 mg/kg dsRNA, 2.6 mg/kg dsRNA, 2.7 mg/kg dsRNA, 2.8 mg/kgdsRNA, 2.9 mg/kg dsRNA, 3.0 mg/kg dsRNA, 3.1 mg/kg dsRNA, 3.2 mg/kgdsRNA, 3.3 mg/kg dsRNA, 3.4 mg/kg dsRNA, 3.5 mg/kg dsRNA, 3.6 mg/kgdsRNA, 3.7 mg/kg dsRNA, 3.8 mg/kg dsRNA, 3.9 mg/kg dsRNA, 4.0 mg/kgdsRNA, 4.1 mg/kg dsRNA, 4.2 mg/kg dsRNA, 4.3 mg/kg dsRNA, 4.4 mg/kgdsRNA, 4.5 mg/kg dsRNA, 4.6 mg/kg dsRNA, 4.7 mg/kg dsRNA, 4.8 mg/kgdsRNA, 4.9 mg/kg dsRNA, 5.0 mg/kg dsRNA, 5.1 mg/kg dsRNA, 5.2 mg/kgdsRNA, 5.3 mg/kg dsRNA, 5.4 mg/kg dsRNA, 5.5 mg/kg dsRNA, 5.6 mg/kgdsRNA, 5.7 mg/kg dsRNA, 5.8 mg/kg dsRNA, 5.9 mg/kg dsRNA, 6.0 mg/kgdsRNA, 6.1 mg/kg dsRNA, 6.2 mg/kg dsRNA, 6.3 mg/kg dsRNA, 6.4 mg/kgdsRNA, 6.5 mg/kg dsRNA, 6.6 mg/kg dsRNA, 6.7 mg/kg dsRNA, 6.8 mg/kgdsRNA, 6.9 mg/kg dsRNA, 7.0 mg/kg dsRNA, 7.1 mg/kg dsRNA, 7.2 mg/kgdsRNA, 7.3 mg/kg dsRNA, 7.4 mg/kg dsRNA, 7.5 mg/kg dsRNA, 7.6 mg/kgdsRNA, 7.7 mg/kg dsRNA, 7.8 mg/kg dsRNA, 7.9 mg/kg dsRNA, 8.0 mg/kgdsRNA, 8.1 mg/kg dsRNA, 8.2 mg/kg dsRNA, 8.3 mg/kg dsRNA, 8.4 mg/kgdsRNA, 8.5 mg/kg dsRNA, 8.6 mg/kg dsRNA, 8.7 mg/kg dsRNA, 8.8 mg/kgdsRNA, 8.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 9.1 mg/kg dsRNA, 9.2 mg/kgdsRNA, 9.3 mg/kg dsRNA, 9.4 mg/kg dsRNA, 9.5 mg/kg dsRNA, 9.6 mg/kgdsRNA, 9.7 mg/kg dsRNA, 9.8 mg/kg dsRNA, 9.9 mg/kg dsRNA, 9.0 mg/kgdsRNA, 10 mg/kg dsRNA, 15 mg/kg dsRNA, 20 mg/kg dsRNA, 25 mg/kg dsRNA,30 mg/kg dsRNA, 35 mg/kg dsRNA, 40 mg/kg dsRNA, 45 mg/kg dsRNA, or about50 mg/kg dsRNA. Values and ranges intermediate to the recited values arealso intended to be part of this invention.

In certain embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and a lipid, subjects can beadministered a therapeutic amount of iRNA, such as about 0.01 mg/kg toabout 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg toabout 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg toabout 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg toabout 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg toabout 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg toabout 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg toabout 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about 2.5mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg,about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In other embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and an N-acetylgalactosamine,subjects can be administered a therapeutic amount of iRNA, such as adose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10to about 20 mg/kg, or about 15 to about 20 mg/kg. In one embodiment,when a composition of the invention comprises a dsRNA as describedherein and an N-acetylgalactosamine, subjects can be administered atherapeutic amount of about 10 to about 30 mg/kg of dsRNA. Values andranges intermediate to the recited values are also intended to be partof this invention.

For example, subjects can be administered a therapeutic amount of iRNA,such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25,25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

The iRNA can be administered by intravenous infusion over a period oftime, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, and 21, 22, 23, 24, or about a 25 minute period. Theadministration may be repeated, for example, on a regular basis, such asweekly, biweekly (i.e., every two weeks) for one month, two months,three months, four months or longer. After an initial treatment regimen,the treatments can be administered on a less frequent basis. Forexample, after administration weekly or biweekly for three months,administration can be repeated once per month, for six months or a yearor longer.

In one embodiment, the present invention provides methods for treating asubject suffering from a bleeding disorder, e.g., a hemophilia, bysubcutaneously administering to said subject compound AD-57213 at acumulative weekly dose of about 0.5 mg/kg to about 5 mg/kg, or about 1mg/kg to about 3 mg/kg.

In one embodiment, the methods may include subcutaneously administeringto the subject a cumulative weekly dose of about 0.5 mg/kg. For example,in one embodiment, the methods may include administering to the subjecta cumulative weekly dose of about 0.5 mg/kg as 0.5.mg/kg every week. Inanother embodiment, the methods may include administering to the subjecta cumulative weekly dose of about 0.5 mg/kg as 1 mg/kg every two weeks.

In another embodiment, the methods may include subcutaneouslyadministering to the subject a cumulative weekly dose of about 1.5mg/kg. For example, in one embodiment, the methods may includeadministering to the subject a cumulative weekly dose of about 1.5 mg/kgas 1.5.mg/kg every week. In another embodiment, the methods may includeadministering to the subject a cumulative weekly dose of about 1.5 mg/kgas 3 mg/kg every two weeks.

In another embodiment, the methods may include subcutaneouslyadministering to the subject a cumulative weekly dose of about 2 mg/kg.For example, in one embodiment, the methods may include administering tothe subject a cumulative weekly dose of about 2 mg/kg as 2 mg/kg everyweek. In another embodiment, the methods may include administering tothe subject a cumulative weekly dose of about 2 mg/kg as 4 mg/kg everytwo weeks.

In yet another embodiment, the methods may include subcutaneouslyadministering to the subject a cumulative weekly dose of about 3 mg/kg.For example, in one embodiment, the methods may include administering tothe subject a cumulative weekly dose of about 3 mg/kg as 3 mg/kg everyweek. In another embodiment, the methods may include administering tothe subject a cumulative weekly dose of about 3 mg/kg as 6 mg/kg everytwo weeks.

In another embodiment, the present invention provides methods forpreventing in a subject at least one symptom of a bleeding disorder,e.g., a hemophilia, by subcutaneously administering to the subjectcompound AD-57213 at a cumulative weekly dose of about 0.5 mg/kg toabout 5 mg/kg or about 1 mg/kg to about 3 mg/kg.

In one embodiment, the methods may include subcutaneously administeringto the subject a cumulative weekly dose of about 0.5 mg/kg. For example,in one embodiment, the methods may include administering to the subjecta cumulative weekly dose of about 0.5 mg/kg as 0.5.mg/kg every week. Inanother embodiment, the methods may include administering to the subjecta cumulative weekly dose of about 0.5 mg/kg as 1 mg/kg every two weeks.

In another embodiment, the methods may include subcutaneouslyadministering to the subject a cumulative weekly dose of about 1.5mg/kg. For example, in one embodiment, the methods may includeadministering to the subject a cumulative weekly dose of about 1.5 mg/kgas 1.5.mg/kg every week. In another embodiment, the methods may includeadministering to the subject a cumulative weekly dose of about 1.5 mg/kgas 3 mg/kg every two weeks.

In another embodiment, the methods may include subcutaneouslyadministering to the subject a cumulative weekly dose of about 2 mg/kg.For example, in one embodiment, the methods may include administering tothe subject a cumulative weekly dose of about 2 mg/kg as 2 mg/kg everyweek. In another embodiment, the methods may include administering tothe subject a cumulative weekly dose of about 2 mg/kg as 4 mg/kg everytwo weeks.

In yet another embodiment, the methods may include subcutaneouslyadministering to the subject a cumulative weekly dose of about 3 mg/kg.For example, in one embodiment, the methods may include administering tothe subject a cumulative weekly dose of about 3 mg/kg as 3 mg/kg everyweek. In another embodiment, the methods may include administering tothe subject a cumulative weekly dose of about 3 mg/kg as 6 mg/kg everytwo weeks.

Administration of the iRNA can reduce Serpinc1 levels, e.g., in a cell,tissue, blood, urine or other compartment of the patient by at leastabout 5%, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, or at least about 99% or more.

In one embodiment, the treatment and/or preventive methods includesubcutaneously administering to a subject compound AD-57213 at a dosesufficient to inhibit reduce Serpinc1 levels, e.g., in a cell, tissue,blood, urine or other compartment of the patient by at least about byabout 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 69, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, or about 80%.

Before administration of a full dose of the iRNA, patients can beadministered a smaller dose, such as a 5% infusion reaction, andmonitored for adverse effects, such as an allergic reaction. In anotherexample, the patient can be monitored for unwanted immunostimulatoryeffects, such as increased cytokine (e.g., TNF-alpha or INF-alpha)levels.

Owing to the inhibitory effects on Serpinc1 expression, a compositionaccording to the invention or a pharmaceutical composition preparedtherefrom can enhance the quality of life.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the iRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES Example 1 iRNA Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent can be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

Transcripts

siRNA design was carried out to identify siRNAs targeting human, rhesus(Macaca mulatta), dog, mouse, and rat SERPINC1 transcripts annotated inthe NCBI Gene database (http://www.ncbi.nlm.nih.gov/gene/). Design usedthe following transcripts from the NCBI RefSeq collection:Human—NM_(—)000488.2, NM_(—)000488.3; Rhesus—NM_(—)001104583.1;Dog—XM_(—)856414.1; Mouse—NM_(—)080844.4; Rat—NM_(—)001012027.1. Due tohigh primate/canine/rodent sequence divergence, siRNA duplexes weredesigned in several separate batches, including but not limited tobatches containing duplexes matching human and rhesus transcripts only;human, rhesus, and dog transcripts only; human, rhesus, mouse, and rattranscripts only; and mouse and rat transcripts only. All siRNA duplexeswere designed that shared 100% identity the listed human transcript andother species transcripts considered in each design batch (above).

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from eachsequence. Candidate 19mers were then selected that lacked repeats longerthan 7 nucleotides. These 874 candidate human/rhesus, 67human/rhesus/dog, 103 human/rhesus/mouse/rat, and 569 mouse/rat siRNAswere used in comprehensive searches against the appropriatetranscriptomes (defined as the set of NM_(—) and XM_(—) records withinthe human, rhesus, dog, mouse, or rat NCBI Refseq sets) using anexhaustive “brute-force” algorithm implemented in the python script‘BruteForce.py’. The script next parsed the transcript-oligo alignmentsto generate a score based on the position and number of mismatchesbetween the siRNA and any potential ‘off-target’ transcript. Theoff-target score is weighted to emphasize differences in the ‘seed’region of siRNAs, in positions 2-9 from the 5′-end of the molecule.

Each oligo-transcript pair from the brute-force search was given amismatch score by summing the individual mismatch scores; mismatches inthe position 2-9 were counted as 2.8, mismatches in the cleavage sitepositions 10-11 were counted as 1.2, and mismatches in region 12-19counted as 1.0. An additional off-target prediction was carried out bycomparing the frequency of heptamers and octomers derived from 3distinct, seed-derived hexamers of each oligo. The hexamers frompositions 2-7 relative to the 5′ start were used to create 2 heptamersand one octamer. ‘Heptamer1’ was created by adding a 3′-A to thehexamer; heptamer2 was created by adding a 5′-A to the hexamer; theoctomer was created by adding an A to both 5′- and 3′-ends of thehexamer. The frequency of octamers and heptamers in the human, rhesus,mouse, or rat 3′-UTRome (defined as the subsequence of the transcriptomefrom NCBI's Refseq database where the end of the coding region, the‘CDS’, is clearly defined) was pre-calculated. The octamer frequency wasnormalized to the heptamer frequency using the median value from therange of octamer frequencies. A ‘mirSeedScore’ was then calculated bycalculating the sum of ((3× normalized octamer count)+(2× heptamer2count)+(1× heptamer1 count)).

Both siRNAs strands were assigned to a category of specificity accordingto the calculated scores: a score above 3 qualifies as highly specific,equal to 3 as specific and between 2.2 and 2.8 as moderately specific.The duplexes were sorted by the specificity of the antisense strand andthose duplexes whose antisense oligos lacked GC at the first position,lacked G at both positions 13 and 14, and had 3 or more Us or As in theseed region were selected.

siRNA Sequence Selection

A total of 66 sense and 66 antisense derived human/rhesus, 6 sense and 6antisense derived human/rhesus/mouse, 12 human/rhesus/mouse/rat, and 21sense and 21 antisense derived mouse/rat siRNA oligos were synthesizedand formed into duplexes. A detailed list of Sepinc1 sense and antisensestrand sequences is shown in Tables 3 and 4.

siRNA Synthesis

I. General Small and Medium Scale RNA Synthesis Procedure

RNA oligonucleotides were synthesized at scales between 0.2-500 μmolusing commercially available5′-O-(4,4′-dimethoxytrityl)-2′-O-t-butyldimethylsilyl-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramiditemonomers of uridine, 4-N-acetylcytidine, 6-N-benzoyladenosine and2-N-isobutyrylguanosine and the corresponding 2′-O-methyl and 2′-fluorophosphoramidites according to standard solid phase oligonucleotidesynthesis protocols. The amidite solutions were prepared at 0.1-0.15 Mconcentration and 5-ethylthio-1H-tetrazole (0.25-0.6 M in acetonitrile)was used as the activator. Phosphorothioate backbone modifications wereintroduced during synthesis using 0.2 M phenylacetyl disulfide (PADS) inlutidine:acetonitrile (1:1) (v;v) or 0.1 M 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) in pyridine for the oxidationstep. After completion of synthesis, the sequences were cleaved from thesolid support and deprotected using methylamine followed bytriethylamine.3HF to remove any 2′-O-t-butyldimethylsilyl protectinggroups present.

For synthesis scales between 5-500 μmol and fully 2′ modified sequences(2′-fluoro and/or 2′-O-methyl or combinations thereof) theoligonucleotides where deprotected using 3:1 (v/v) ethanol andconcentrated (28-32%) aqueous ammonia either at 35° C. 16 h or 55° C.for 5.5 h. Prior to ammonia deprotection the oligonucleotides wheretreated with 0.5 M piperidine in acetonitrile for 20 min on the solidsupport. The crude oligonucleotides were analyzed by LC-MS andanion-exchange HPLC (IEX-HPLC). Purification of the oligonucleotides wascarried out by IEX HPLC using: 20 mM phosphate, 10%-15% ACN, pH=8.5(buffer A) and 20 mM phosphate, 10%-15% ACN, 1 M NaBr, pH=8.5 (bufferB). Fractions were analyzed for purity by analytical HPLC. Theproduct-containing fractions with suitable purity were pooled andconcentrated on a rotary evaporator prior to desalting. The samples weredesalted by size exclusion chromatography and lyophilized to dryness.Equal molar amounts of sense and antisense strands were annealed in1×PBS buffer to prepare the corresponding siRNA duplexes.

For small scales (0.2-1 μmol), synthesis was performed on a MerMade 192synthesizer in a 96 well format. In case of fully 2′-modified sequences(2′-fluoro and/or 2′-O-methyl or combinations thereof) theoligonucleotides where deprotected using methylamine at room temperaturefor 30-60 min followed by incubation at 60° C. for 30 min or using 3:1(v/v) ethanol and concentrated (28-32%) aqueous ammonia at roomtemperature for 30-60 min followed by incubation at 40° C. for 1.5hours. The crude oligonucleotides were then precipitated in a solutionof acetonitrile:acetone (9:1) and isolated by centrifugation anddecanting the supernatant. The crude oligonucleotide pellet wasre-suspended in 20 mM NaOAc buffer and analyzed by LC-MS and anionexchange HPLC. The crude oligonucleotide sequences were desalted in 96deep well plates on a 5 mL HiTrap Sephadex G25 column (GE Healthcare).In each well about 1.5 mL samples corresponding to an individualsequence was collected. These purified desalted oligonucleotides wereanalyzed by LC-MS and anion exchange chromatography. Duplexes wereprepared by annealing equimolar amounts of sense and antisense sequenceson a Tecan robot. Concentration of duplexes was adjusted to 10 μM in1×PBS buffer.

II. Synthesis of GalNAc-Conjugated Oligonucleotides for In Vivo Analysis

Oligonucleotides conjugated with GalNAc ligand at their 3′-terminus weresynthesized at scales between 0.2-500 μmol using a solid supportpre-loaded with a Y-shaped linker bearing a 4,4′-dimethoxytrityl(DMT)-protected primary hydroxy group for oligonucleotide synthesis anda GalNAc ligand attached through a tether.

For synthesis of GalNAc conjugates in the scales between 5-500 μmol, theabove synthesis protocol for RNA was followed with the followingadaptions: For polystyrene-based synthesis supports 5% dichloroaceticacid in toluene was used for DMT-cleavage during synthesis. Cleavagefrom the support and deprotection was performed as described above.Phosphorothioate-rich sequences (usually >5 phorphorothioates) weresynthesized without removing the final 5′-DMT group (“DMT-on”) and,after cleavage and deprotection as described above, purified by reversephase HPLC using 50 mM ammonium acetate in water (buffer A) and 50 mMammoniumacetate in 80% acetonitrile (buffer B). Fractions were analyzedfor purity by analytical HPLC and/or LC-MS. The product-containingfractions with suitable purity were pooled and concentrated on a rotaryevaporator. The DMT-group was removed using 20%-25% acetic acid in wateruntil completion. The samples were desalted by size exclusionchromatography and lyophilized to dryness. Equal molar amounts of senseand antisense strands were annealed in 1×PBS buffer to prepare thecorresponding siRNA duplexes.

For small scale synthesis of GalNAc conjugates (0.2-1 μmol), includingsequences with multiple phosphorothioate linkages, the protocolsdescribed above for synthesis of RNA or fully 2′-F/2′-OMe-containingsequences on MerMade platform were applied. Synthesis was performed onpre-packed columns containing GalNAc-functionalized controlled poreglass support.

Example 2 In Vitro Screening Cell Culture and Transfections

Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO2 in Eagle's Minimum Essential Medium (ATCC)supplemented with 10% FBS, streptomycin, and glutamine (ATCC) beforebeing released from the plate by trypsinization. For mouse crossreactive duplexes, primary mouse hepatocytes (PMH) were freshly isolatedless than 1 hour prior to transfections and grown in primary hepatocytemedia. For both Hep3B and PMH, transfection was carried out by adding14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well(Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of each siRNAduplex to an individual well in a 96-well plate. The mixture was thenincubated at room temperature for 15 minutes. Eighty μl of completegrowth media without antibiotic containing ˜2×10⁴ Hep3B cells were thenadded to the siRNA mixture. Cells were incubated for 24 hours prior toRNA purification. Single dose experiments were performed at 10 nM and0.1 nM final duplex concentration and dose response experiments weredone using 8×5-fold serial dilutions over the range of 10 nM to 128 pM(see FIGS. 2A and 2B).

Free Uptake Transfection

Five μl of each GalNac conjugated siRNA in PBS was combined with 4×10⁴freshly thawed cryopreserved Cynomolgus monkey hepatocytes resuspendedin 95 μl of In Vitro Gro CP media (In Vitro Technologies-Celsis,Baltimore, Md.) in each well of a 96 well plate. The mixture wasincubated for about 24 hrs at 37° C. in an atmosphere of 5% CO₂. siRNAswere tested at final concentrations of 100 nM, 10 nM and 0.1 nM forefficacy free uptake assays.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12)

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (themixing speed was the same throughout the process). Ten microliters ofmagnetic beads and 80 μl Lysis/Binding Buffer mixture were added to around bottom plate and mixed for 1 minute. Magnetic beads were capturedusing a magnetic stand and the supernatant was removed withoutdisturbing the beads. After removing the supernatant, the lysed cellswere added to the remaining beads and mixed for 5 minutes. Afterremoving the supernatant, magnetic beads were washed 2 times with 150 μlWash Buffer A and mixed for 1 minute. The beads were captured again andthe supernatant was removed. The beads were then washed with 150 μl WashBuffer B, captured and the supernatant was removed. The beads were nextwashed with 150 μl Elution Buffer, captured and the supernatant removed.Finally, the beads were allowed to dry for 2 minutes. After drying, 50μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Thebeads were captured on magnet for 5 minutes. 40 μl of supernatant wasremoved and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O perreaction were added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. for 10 minutes, 37° C. for 120 minutes, 85° C.for 5 seconds, and 4° C. hold.

Real Time PCR

Two μl of cDNA were added to a master mix containing 0.5 μl human GAPDHTaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl human SERPINC1TaqMan probe (Applied Biosystems cat # Hs00892758_m1) for human cells or0.5 μl mouse GAPDH TaqMan Probe (Applied Biosystems Cat #4308313), 0.5μl mouse SERPINC1 TaqMan probe (Applied Biosystems cat # Mm00446573_m1)for mouse cells and 5 μl Lightcycler 480 probe master mix (Roche Cat#04887301001) per well in a 384 well plates. Real time PCR was done inan ABI 7900HT Real Time PCR system (Applied Biosystems) using theΔΔCt(RQ) assay. Each duplex was tested in two independent transfectionsand each transfection was assayed in duplicate, unless otherwise notedin the summary tables.

To calculate relative fold change in Serpinc1 mRNA levels, real timedata were analyzed using the ΔΔCt method and normalized to assaysperformed with cells transfected with 10 nM AD-1955, or mock transfectedcells. IC₅₀s were calculated using a 4 parameter fit model using XLFitand normalized to cells transfected with AD-1955 over the same doserange, or to its own lowest dose. Table 5 shows the results of a singledose screen in Hep3B cells and PMH cells transfected with the indicatediRNAs. Table 6 shows the results of dose response of the indicated iRNAstransfected into Hep3B and PMH cells.

The sense and antisense sequences of AD-1955 are:

SENSE: (SEQ ID NO: 13) cuuAcGcuGAGuAcuucGAdTsdT ANTISENSE:(SEQ ID NO: 14) UCGAAGuACUcAGCGuAAGdTsdT.

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) AAdenosine-3′-phosphate Ab beta-L-adenosine-3′-phosphate Absbeta-L-adenosine-3′-phosphorothioate Af 2′-fluoroadenosine-3′-phosphateAfs 2′-fluoroadenosine-3′-phosphorothioate Asadenosine-3′-phosphorothioate C cytidine-3′-phosphate Cbbeta-L-cytidine-3′-phosphate Cbs beta-L-cytidine-3′-phosphorothioate Cf2′-fluorocytidine-3′-phosphate Cfs 2′-fluorocytidine-3′-phosphorothioate(Chd) 2′-O-hexadecyl-cytidine-3′-phosphate (Chds)2′-O-hexadecyl-cytidine-3′-phosphorothioate Cscytidine-3′-phosphorothioate G guanosine-3′-phosphate Gbbeta-L-guanosine-3′-phosphate Gbs beta-L-guanosine-3′-phosphorothioateGf 2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate Tb beta-L-thymidine-3′-phosphate Tbsbeta-L-thymidine-3′-phosphorothioate Tf2′-fluoro-5-methyluridine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Ubbeta-L-uridine-3′-phosphate Ubs beta-L-uridine-3′-phosphorothioate Uf2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioate(Uhd) 2′-O-hexadecyl-uridine-3′-phosphate (Uhds)2′-O-hexadecyl-uridine-3′-phosphorothioate Usuridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs2′-O-methylcytidine-3′-phosphorothioate g2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyruridine-3′-phosphorothioate dA 2′-deoxyadenosine-3′-phosphatedAs 2′-deoxyadenosine-3′-phosphorothioate dC2′-deoxycytidine-3′-phosphate dCs 2′-deoxycytidine-3′-phosphorothioatedG 2′-deoxyguanosine-3′-phosphate dGs2′-deoxyguanosine-3′-phosphorothioate dT 2′-deoxythymidine dTs2′-deoxythymidine-3′-phosphorothioate dU 2′-deoxyuridine sphosphorothioate linkage L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinolHyp-(GalNAc-alkyl)3 (Aeo) 2′-O-methoxyethyladenosine-3′-phosphate (Aeos)2′-O-methoxyethyladenosine-3′-phosphorothioate (Geo)2′-O-methoxyethylguanosine-3′-phosphate (Geos)2′-O-methoxyethylguanosine-3′-phosphorothioate (Teo)2′-O-methoxyethyl-5-methyluridine-3′-phosphate (Teos)2′-O-methoxyethyl-5-methyluridine-3′-phosphorothioate (m5Ceo)2′-O-methoxyethyl-5-methylcytidine-3′-phosphate (m5Ceos)2′-O-methoxyethyl-5-methylcytidine-3′-phosphorothioate

TABLE 3 Unmodified Sense and antisense strand sequences ofSerpinc1 dsRNAs (The “Sense Sequence” columnsequences are disclosed as SEQ ID NOS 15-71,respectively, in order of appearance, and the “Antisense Sequence”column sequences are disclosed as SEQ ID NOS 72-128, respectively,in order of appearance) Duplex Sense Sense Antisense Antisense Name NameSequence Name Sequence AD-50475.1-UM A-104633.1 CCCUGUGGACAUCUGCACAA-104634.1 UGUGCAGAUGUCCACAGGG AD-50476.1-UM A-104649.1CUACCACUUUCUAUCAGCA A-104650.1 UGCUGAUAGAAAGUGGUAG AD-50477.1-UMA-104665.1 CUAUCGAAAAGCCAACAAA A-104666.1 UUUGUUGGCUUUUCGAUAGAD-50478.1-UM A-104681.1 GGACUUCAAGGAAAAUGCA A-104682.1UGCAUUUUCCUUGAAGUCC AD-50479.1-UM A-104697.1 GUUAACACCAUUUACUUCAA-104698.1 UGAAGUAAAUGGUGUUAAC AD-50480.1-UM A-104713.1CCUGGUUUUUAUAAGAGAA A-104714.1 UUCUCUUAUAAAAACCAGG AD-50481.1-UMA-104635.1 GACAUUCCCAUGAAUCCCA A-104636.1 UGGGAUUCAUGGGAAUGUCAD-50482.1-UM A-104651.1 CACCUGGCAGAUUCCAAGA A-104652.1UCUUGGAAUCUGCCAGGUG AD-50483.1-UM A-104667.1 CGAAAAGCCAACAAAUCCUA-104668.1 AGGAUUUGUUGGCUUUUCG AD-50484.1-UM A-104683.1GAAAAUGCAGAGCAAUCCA A-104684.1 UGGAUUGCUCUGCAUUUUC AD-50485.1-UMA-104699.1 GGCCUGUGGAAGUCAAAGU A-104700.1 ACUUUGACUUCCACAGGCCAD-50486.1-UM A-104715.1 GAAGUUCCUCUGAACACUA A-104716.1UAGUGUUCAGAGGAACUUC AD-50487.1-UM A-104637.1 CCAUGAAUCCCAUGUGCAUA-104638.1 AUGCACAUGGGAUUCAUGG AD-50488.1-UM A-104653.1CAACUGAUGGAGGUAUUUA A-104654.1 UAAAUACCUCCAUCAGUUG AD-50489.1-UMA-104669.1 CCAAGUUAGUAUCAGCCAA A-104670.1 UUGGCUGAUACUAACUUGGAD-50490.1-UM A-104685.1 CGGCCAUCAACAAAUGGGU A-104686.1ACCCAUUUGUUGAUGGCCG AD-50491.1-UM A-104701.1 GAGGACGGCUUCAGUUUGAA-104702.1 UCAAACUGAAGCCGUCCUC AD-50492.1-UM A-104717.1CCUCUGAACACUAUUAUCU A-104718.1 AGAUAAUAGUGUUCAGAGG AD-50493.1-UMA-104639.1 CAUGAAUCCCAUGUGCAUU A-104640.1 AAUGCACAUGGGAUUCAUGAD-50494.1-UM A-104655.1 GAUGGAGGUAUUUAAGUUU A-104656.1AAACUUAAAUACCUCCAUC AD-50495.1 UM A-104671.1 GUAUCAGCCAAUCGCCUUUA-104672.1 AAAGGCGAUUGGCUGAUAC AD-50496.1-UM A-104687.1GGGUGUCCAAUAAGACCGA A-104688.1 UCGGUCUUAUUGGACACCC AD-50497.1-UMA-104703.1 CAGCCCUGAAAAGUCCAAA A-104704.1 UUUGGACUUUUCAGGGCUGAD-50498.1-UM A-104641.1 CCCAUGUGCAUUUACCGCU A-104642.1AGCGGUAAAUGCACAUGGG AD-50499.1-UM A-104657.1 GUAUUUAAGUUUGACACCAA-104658.1 UGGUGUCAAACUUAAAUAC AD-50500.1-UM A-104673.1GACAAAUCCCUUACCUUCA A-104674.1 UGAAGGUAAGGGAUUUGUC AD-50501.1-UMA-104689.1 CUGUUCUGGUGCUGGUUAA A-104690.1 UUAACCAGCACCAGAACAGAD-50502.1 UM A-104705.1 CCAAACUCCCAGGUAUUGU A-104706.1ACAAUACCUGGGAGUUUGG AD-50503.1-UM A-104643.1 CCCGCUUUGCUACCACUUUA-104644.1 AAAGUGGUAGCAAAGCGGG AD-50505.1-UM A-104675.1CUUACCUUCAAUGAGACCU A-104676.1 AGGUCUCAUUGAAGGUAAG AD-50506.1-UMA-104691.1 CUGGUGCUGGUUAACACCA A-104692.1 UGGUGUUAACCAGCACCAGAD-50507.1-UM A-104707.1 CAAACUCCCAGGUAUUGUU A-104708.1AACAAUACCUGGGAGUUUG AD-50508.1-UM A-104645.1 GCUUUGCUACCACUUUCUAA-104646.1 UAGAAAGUGGUAGCAAAGC AD-50510.1-UM A-104677.1CCUACCAGGACAUCAGUGA A-104678.1 UCACUGAUGUCCUGGUAGG AD-50511.1-UMA-104693.1 GGUGCUGGUUAACACCAUU A-104694.1 AAUGGUGUUAACCAGCACCAD-50512.1-UM A-104709.1 GCCGUUCGCUAAACCCCAA A-104710.1UUGGGGUUUAGCGAACGGC AD-50515.1-UM A-104679.1 GGACAUCAGUGAGUUGGUAA-104680.1 UACCAACUCACUGAUGUCC AD-50516.1-UM A-104695.1GUGCUGGUUAACACCAUUU A-104696.1 AAAUGGUGUUAACCAGCAC AD-50517.1-UMA-104711.1 GCCUUUCCUGGUUUUUAUA A-104712.1 UAUAAAAACCAGGAAAGGCAD-50518.1-UM A-104719.1 CUUUUGCUAUGACCAAGCU A-104720.1AGCUUGGUCAUAGCAAAAG AD-50523.1-UM A-104721.1 UGUACCAGGAAGGCAAGUUA-104722.1 AACUUGCCUUCCUGGUACA AD-50528.1-UM A-104723.1ACUAUUAUCUUCAUGGGCA A-104724.1 UGCCCAUGAAGAUAAUAGU AD-50540.1-UMA-104729.1 UCAUGGGCAGAGUAGCCAA A-104730.1 UUGGCUACUCUGCCCAUGAAD-50539.1-UM A-104785.1 CCAUUUACUUCAAGGGCCU A-104786.1AGGCCCUUGAAGUAAAUGG AD-50544.1-UM A-104787.1 UACUUCAAGGGCCUGUGGAA-104788.1 UCCACAGGCCCUUGAAGUA AD-50549.1-UM A-104789.1ACUUCAAGGGCCUGUGGAA A-104790.1 UUCCACAGGCCCUUGAAGU AD-50514.1-UMA-104663.1 CGACUCUAUCGAAAAGCCA A-104664.1 UGGCUUUUCGAUAGAGUCGAD-50522.1-UM A-104779.1 AACUGCCGACUCUAUCGAA A-104780.1UUCGAUAGAGUCGGCAGUU AD-50527.1-UM A-104781.1 ACUGCCGACUCUAUCGAAAA-104782.1 UUUCGAUAGAGUCGGCAGU AD-50531.1-UM A-104739.1GACUCUAUCGAAAAGCCAA A-104740.1 UUGGCUUUUCGAUAGAGUC AD-50534.1-UMA-104769.1 UCUUCUUUGCCAAACUGAA A-104770.1 UUCAGUUUGGCAAAGAAGAAD-50538.1-UM A-104771.1 UGCCAAACUGAACUGCCGA A-104772.1UCGGCAGUUCAGUUUGGCA AD-50543.1-UM A-104773.1 CCAAACUGAACUGCCGACUA-104774.1 AGUCGGCAGUUCAGUUUGG AD-50553.1-UM A-104777.1ACUGAACUGCCGACUCUAU A-104778.1 AUAGAGUCGGCAGUUCAGU AD-50504.1-UMA-104659.1 GAACUGCCGACUCUAUCGA A-104660.1 UCGAUAGAGUCGGCAGUUCAD-50509.1-UM A-104661.1 CUGCCGACUCUAUCGAAAA A-104662.1UUUUCGAUAGAGUCGGCAG AD-50529.1-UM A-104751.1 CUGGUUAACACCAUUUACUA-104752.1 AGUAAAUGGUGUUAACCAG

TABLE 4 Modified Sense and antisense strand sequences of Serpinc1dsRNAs (The “Sense Sequence” column sequences aredisclosed as SEQ ID NOS 129-185, respectively, in orderof appearance, and the “Antisense Sequence” columnsequences are disclosed as SEQ ID NOS 186-242,respectively, in order of appearance) Duplex Sense Sense AntisenseAntisense Name Name Sequence Name Sequence AD-50475.1 A-104633.1cccuGuGGAcAucuGcAcAdTsdT A-104634.1 UGUGcAGAUGUCcAcAGGGdTsdT AD-50476.1A-104649.1 cuAccAcuuucuAucAGcAdTsdT A-104650.1 UGCUGAuAGAAAGUGGuAGdTsdTAD-50477.1 A-104665.1 cuAucGAAAAGccAAcAAAdTsdT A-104666.1UUUGUUGGCUUUUCGAuAGdTsdT AD-50478.1 A-104681.1 GGAcuucAAGGAAAAuGcAdTsdTA-104682.1 UGcAUUUUCCUUGAAGUCCdTsdT AD-50479.1 A-104697.1GuuAAcAccAuuuAcuucAdTsdT A-104698.1 UGAAGuAAAUGGUGUuAACdTsdT AD-50480.1A-104713.1 ccuGGuuuuuAuAAGAGAAdTsdT A-104714.1 UUCUCUuAuAAAAACcAGGdTsdTAD-50481.1 A-104635.1 GAcAuucccAuGAAucccAdTsdT A-104636.1UGGGAUUcAUGGGAAUGUCdTsdT AD-50482.1 A-104651.1 cAccuGGcAGAuuccAAGAdTsdTA-104652.1 UCUUGGAAUCUGCcAGGUGdTsdT AD-50483.1 A-104667.1cGAAAAGccAAcAAAuccudTsdT A-104668.1 AGGAUUUGUUGGCUUUUCGdTsdT AD-50484.1A-104683.1 GAAAAuGcAGAGcAAuccAdTsdT A-104684.1 UGGAUUGCUCUGcAUUUUCdTsdTAD-50485.1 A-104699.1 GGccuGuGGAAGucAAAGudTsdT A-104700.1ACUUUGACUUCcAcAGGCCdTsdT AD-50486.1 A-104715.1 GAAGuuccucuGAAcAcuAdTsdTA-104716.1 uAGUGUUcAGAGGAACUUCdTsdT AD-50487.1 A-104637.1ccAuGAAucccAuGuGcAudTsdT A-104638.1 AUGcAcAUGGGAUUcAUGGdTsdT AD-50488.1A-104653.1 cAAcuGAuGGAGGuAuuuAdTsdT A-104654.1 uAAAuACCUCcAUcAGUUGdTsdTAD-50489.1 A-104669.1 ccAAGuuAGuAucAGccAAdTsdT A-104670.1UUGGCUGAuACuAACUUGGdTsdT AD-50490.1 A-104685.1 cGGccAucAAcAAAuGGGudTsdTA-104686.1 ACCcAUUUGUUGAUGGCCGdTsdT AD-50491.1 A-104701.1GAGGAcGGcuucAGuuuGAdTsdT A-104702.1 UcAAACUGAAGCCGUCCUCdTsdT AD-50492.1A-104717.1 ccucuGAAcAcuAuuAucudTsdT A-104718.1 AGAuAAuAGUGUUcAGAGGdTsdTAD-50493.1 A-104639.1 cAuGAAucccAuGuGcAuudTsdT A-104640.1AAUGcAcAUGGGAUUcAUGdTsdT AD-50494.1 A-104655.1 GAuGGAGGuAuuuAAGuuudTsdTA-104656.1 AAACUuAAAuACCUCcAUCdTsdT AD-50495.1 A-104671.1GuAucAGccAAucGccuuudTsdT A-104672.1 AAAGGCGAUUGGCUGAuACdTsdT AD-50496.1A-104687.1 GGGuGuccAAuAAGAccGAdTsdT A-104688.1 UCGGUCUuAUUGGAcACCCdTsdTAD-50497.1 A-104703.1 cAGcccuGAAAAGuccAAAdTsdT A-104704.1UUUGGACUUUUcAGGGCUGdTsdT AD-50498.1 A-104641.1 cccAuGuGcAuuuAccGcudTsdTA-104642.1 AGCGGuAAAUGcAcAUGGGdTsdT AD-50499.1 A-104657.1GuAuuuAAGuuuGAcAccAdTsdT A-104658.1 UGGUGUcAAACUuAAAuACdTsdT AD-50500.1A-104673.1 GAcAAAucccuuAccuucAdTsdT A-104674.1 UGAAGGuAAGGGAUUUGUCdTsdTAD-50501.1 A-104689.1 cuGuucuGGuGcuGGuuAAdTsdT A-104690.1UuAACcAGcACcAGAAcAGdTsdT AD-50502.1 A-104705.1 ccAAAcucccAGGuAuuGudTsdTA-104706.1 AcAAuACCUGGGAGUUUGGdTsdT AD-50503.1 A-104643.1cccGcuuuGcuAccAcuuudTsdT A-104644.1 AAAGUGGuAGcAAAGCGGGdTsdT AD-50505.1A-104675.1 cuuAccuucAAuGAGAccudTsdT A-104676.1 AGGUCUcAUUGAAGGuAAGdTsdTAD-50506.1 A-104691.1 cuGGuGcuGGuuAAcAccAdTsdT A-104692.1UGGUGUuAACcAGcACcAGdTsdT AD-50507.1 A-104707.1 cAAAcucccAGGuAuuGuudTsdTA-104708.1 AAcAAuACCUGGGAGUUUGdTsdT AD-50508.1 A-104645.1GcuuuGcuAccAcuuucuAdTsdT A-104646.1 uAGAAAGUGGuAGcAAAGCdTsdT AD-50510.1A-104677.1 ccuAccAGGAcAucAGuGAdTsdT A-104678.1 UcACUGAUGUCCUGGuAGGdTsdTAD-50511.1 A-104693.1 GGuGcuGGuuAAcAccAuudTsdT A-104694.1AAUGGUGUuAACcAGcACCdTsdT AD-50512.1 A-104709.1 GccGuucGcuAAAccccAAdTsdTA-104710.1 UUGGGGUUuAGCGAACGGCdTsdT AD-50515.1 A-104679.1GGAcAucAGuGAGuuGGuAdTsdT A-104680.1 uACcAACUcACUGAUGUCCdTsdT AD-50516.1A-104695.1 GuGcuGGuuAAcAccAuuudTsdT A-104696.1 AAAUGGUGUuAACcAGcACdTsdTAD-50517.1 A-104711.1 GccuuuccuGGuuuuuAuAdTsdT A-104712.1uAuAAAAACcAGGAAAGGCdTsdT AD-50518.1 A-104719.1 cuuuuGcuAuGAccAAGcudTsdTA-104720.1 AGCUUGGUcAuAGcAAAAGdTsdT AD-50523.1 A-104721.1uGuAccAGGAAGGcAAGuudTsdT A-104722.1 AACUUGCCUUCCUGGuAcAdTsdT AD-50528.1A-104723.1 AcuAuuAucuucAuGGGcAdTsdT A-104724.1 UGCCcAUGAAGAuAAuAGUdTsdTAD-50540.1 A-104729.1 ucAuGGGcAGAGuAGccAAdTsdT A-104730.1UUGGCuACUCUGCCcAUGAdTsdT AD-50539.1 A-104785.1 ccAuuuAcuucAAGGGccudTsdTA-104786.1 AGGCCCUUGAAGuAAAUGGdTsdT AD-50544.1 A-104787.1uAcuucAAGGGccuGuGGAdTsdT A-104788.1 UCcAcAGGCCCUUGAAGuAdTsdT AD-50549.1A-104789.1 AcuucAAGGGccuGuGGAAdTsdT A-104790.1 UUCcAcAGGCCCUUGAAGUdTsdTAD-50514.1 A-104663.1 cGAcucuAucGAAAAGccAdTsdT A-104664.1UGGCUUUUCGAuAGAGUCGdTsdT AD-50522.1 A-104779.1 AAcuGccGAcucuAucGAAdTsdTA-104780.1 UUCGAuAGAGUCGGcAGUUdTsdT AD-50527.1 A-104781.1AcuGccGAcucuAucGAAAdTsdT A-104782.1 UUUCGAuAGAGUCGGcAGUdTsdT AD-50531.1A-104739.1 GAcucuAucGAAAAGccAAdTsdT A-104740.1 UUGGCUUUUCGAuAGAGUCdTsdTAD-50534.1 A-104769.1 ucuucuuuGccAAAcuGAAdTsdT A-104770.1UUcAGUUUGGcAAAGAAGAdTsdT AD-50538.1 A-104771.1 uGccAAAcuGAAcuGccGAdTsdTA-104772.1 UCGGcAGUUcAGUUUGGcAdTsdT AD-50543.1 A-104773.1ccAAAcuGAAcuGccGAcudTsdT A-104774.1 AGUCGGcAGUUcAGUUUGGdTsdT AD-50553.1A-104777.1 AcuGAAcuGccGAcucuAudTsdT A-104778.1 AuAGAGUCGGcAGUUcAGUdTsdTAD-50504.1 A-104659.1 GAAcuGccGAcucuAucGAdTsdT A-104660.1UCGAuAGAGUCGGcAGUUCdTsdT AD-50509.1 A-104661.1 cuGccGAcucuAucGAAAAdTsdTA-104662.1 UUUUCGAuAGAGUCGGcAGdTsdT AD-50529.1 A-104751.1cuGGuuAAcAccAuuuAcudTsdT A-104752.1 AGuAAAUGGUGUuAACcAGdTsdT

TABLE 5¹ Serpinc1 single dose screen Human (Hep3B) Mouse (PMH) DuplexName 10 nM Ave 0.1 nM Ave 10 nM Ave 0.1 nM Ave AD-50475.1 0.11 0.21AD-50476.1 0.08 0.43 AD-50477.1 0.10 0.10 AD-50478.1 0.12 0.36AD-50479.1 0.24 0.84 AD-50480.1 0.31 0.73 AD-50481.1 0.74 1.12AD-50482.1 0.61 0.89 AD-50483.1 0.07 0.14 AD-50484.1 0.12 0.33AD-50485.1 0.58 1.18 AD-50486.1 0.79 0.94 AD-50487.1 0.05 0.09AD-50488.1 0.83 1.07 AD-50489.1 0.09 0.28 AD-50490.1 0.04 0.78AD-50491.1 0.19 0.77 AD-50492.1 0.16 0.84 AD-50493.1 0.17 0.55AD-50494.1 0.16 0.59 AD-50495.1 0.08 0.13 AD-50496.1 0.57 0.94AD-50497.1 0.85 1.15 AD-50498.1 0.16 1.02 AD-50499.1 0.10 0.21AD-50500.1 0.22 0.58 AD-50501.1 0.10 0.32 AD-50502.1 0.76 1.07AD-50503.1 0.08 0.47 AD-50505.1 0.74 0.77 AD-50506.1 0.85 0.89AD-50507.1 0.03 0.37 AD-50508.1 0.16 0.97 AD-50510.1 0.09 0.89AD-50511.1 0.15 0.71 AD-50512.1 0.88 1.19 AD-50515.1 0.13 0.49AD-50516.1 0.85 0.95 AD-50517.1 0.14 0.59 AD-50518.1 0.36 1.05AD-50523.1 0.03 0.66 AD-50528.1 0.04 0.27 AD-50540.1 0.14 0.37AD-50539.1 0.09 0.46 0.39 1.10 AD-50544.1 0.23 0.75 0.36 1.07 AD-50549.10.10 0.19 0.17 0.71 AD-50514.1 0.12 0.48 0.19 0.95 AD-50522.1 0.61 1.020.46 1.32 AD-50527.1 0.06 0.15 0.08 0.45 AD-50531.1 0.09 0.47 0.24 1.04AD-50534.1 0.05 0.10 0.11 0.55 AD-50538.1 0.61 0.86 0.79 1.23 AD-50543.10.40 1.04 0.49 1.23 AD-50553.1 0.40 0.93 0.72 1.25 AD-50504.1 ND ND 0.921.25 AD-50509.1 ND ND 0.12 0.37 AD-50529.1 ND ND 0.23 0.47 ¹Modified.

TABLE 6 Serpinc1 IC₅₀ Data Hep3B IC₅₀ PMH IC₅₀ Duplex Name (nM) (nM)AD-50487.1 0.003 — AD-50477.1 0.006 — AD-50483.1 0.011 — AD-50475.10.011 — AD-50495.1 0.017 — AD-50476.1 0.026 — AD-50499.1 0.027 —AD-50478.1 0.028 — AD-50489.1 0.029 — AD-50501.1 0.045 — AD-50507.10.052 — AD-50484.1 0.081 — AD-50515.1 0.185 — AD-50540.1 0.023 —AD-50528.1 0.056 — AD-50549.1 0.053 ND AD-50539.1 0.170 ND AD-50534.10.007 ND AD-50527.1 0.028 ND AD-50514.1 0.085 ND AD-50527.1 ND 0.019AD-50534.1 ND 0.011 AD-50509.1 ND 0.006 AD-50529.1 ND 0.021

A subset of siRNAs were also synthesized with 2′-OMe modifications, andduplexes of these siRNAs in lipofectamine formulations were used totransfect Hep3B cells. The results of the single dose screen of themodified duplexes are shown in Table 7.

TABLE 7 Lead Optimization (2′-OMe variants) Parent Duplex ID Ave 1 nMAve 0.1 nM Ave 0.01 nM AD-50477 AD-50477.1 0.22 0.33 0.53 AD-50477AD-55025.1 0.29 0.68 0.86 AD-50477 AD-55031.1 0.42 0.74 0.93 AD-50477AD-55037.1 0.52 0.73 0.95 AD-50477 AD-55043.1 0.45 0.70 0.94 AD-50477AD-55049.1 0.24 0.47 0.95 AD-50477 AD-55055.1 0.37 0.68 0.99 AD-50477AD-55061.1 0.43 0.66 0.85 AD-50477 AD-55067.1 0.56 0.72 0.92 AD-50477AD-55026.1 0.28 0.59 0.87 AD-50477 AD-55032.1 0.49 0.76 0.86 AD-50477AD-55038.1 0.52 0.75 0.93 AD-50477 AD-55044.1 0.84 0.77 1.06 AD-50487AD-50487.1 0.21 0.50 0.76 AD-50487 AD-55050.1 0.24 0.53 0.75 AD-50487AD-55056.1 0.27 0.50 0.84 AD-50487 AD-55062.1 0.30 0.61 0.84 AD-50487AD-55068.1 0.20 0.37 0.66 AD-50487 AD-55027.1 0.18 0.36 0.67 AD-50487AD-55033.1 0.22 0.43 0.70 AD-50487 AD-55039.1 0.19 0.38 0.67 AD-50487AD-55045.1 0.18 0.29 0.57 AD-50487 AD-55051.1 0.17 0.29 0.60 AD-50487AD-55057.1 0.21 0.37 0.65 AD-50487 AD-55063.1 0.19 0.33 0.63 AD-50487AD-55069.1 0.16 0.26 0.51 AD-50509 AD-50509.1 0.15 0.31 0.57 AD-50509AD-55029.1 0.17 0.26 0.49 AD-50509 AD-55028.1 0.17 0.35 0.54 AD-50509AD-55052.1 0.21 0.32 0.59 AD-50509 AD-55035.1 0.19 0.31 0.62 AD-50509AD-55047.1 0.19 0.35 0.66 AD-50509 AD-55058.1 0.21 0.40 0.66 AD-50509AD-55046.1 0.18 0.37 0.66 AD-50509 AD-55070.1 0.17 0.40 0.68 AD-50509AD-55034.1 0.19 0.37 0.69 AD-50509 AD-55041.1 0.20 0.28 0.63 AD-50509AD-55064.1 0.19 0.34 0.65 AD-50509 AD-55040.1 0.18 0.34 0.69 AD-50534AD-50534.1 0.19 0.42 0.83 AD-50534 AD-55053.1 0.24 0.38 0.59 AD-50534AD-55030.1 0.15 0.33 0.64 AD-50534 AD-55054.1 0.18 0.40 0.69 AD-50534AD-55059.1 0.18 0.33 0.56 AD-50534 AD-55036.1 0.22 0.37 0.61 AD-50534AD-55060.1 0.19 0.42 0.62 AD-50534 AD-55071.1 0.29 0.56 0.81 AD-50534AD-55048.1 0.26 0.56 0.83 AD-50534 AD-55066.1 0.30 0.49 0.76 AD-50534AD-55042.1 0.25 0.47 0.79 AD-50534 AD-55065.1 0.24 0.50 0.83

Examples 3-4 Lead Optimization and In Vivo Testing

Table 8 is a detailed list of sequences of duplex siRNAs targetingSerpinc1 that were formulated as a lipid nanoparticle (LNP) (i.e., withMC3) or conjugated to GalNAc for lead optimization and in vivo delivery.

TABLE 8 Sense and antisense strand sequences of Serpinc1 dsRNAsfor lead optimization (The “Sense Sequence” column sequencesare disclosed as SEQ ID NOS 243-510, respectively, inorder of appearance, and the “Antisense Sequence” column sequences are disclosed as SEQ ID NOS 511-778, respectively,in order of appearance) Duplex Sense Sense Antisense Antisense Name NameSequence Name Sequences AD-50477.1 A-104665.1 cuAucGAAAAGccAAcAAAdTsdTA-104666.1 UUUGUUGGCUUUUCGAuAGdTsdT AD-55025.1 A-113301.1cuAucGAAAAGccAAcAAAdTdT A-113302.1 UUUGUUGGCUUUUcGAuAGdTdT AD-55031.1A-113301.2 cuAucGAAAAGccAAcAAAdTdT A-113303.1 UUUGUUGGCUUuUcGAuAGdTdTAD-55037.1 A-113301.3 cuAucGAAAAGccAAcAAAdTdT A-113304.1UUUGUUGGcUUuUcGAuAGdTdT AD-55043.1 A-113301.4 cuAucGAAAAGccAAcAAAdTdTA-113305.1 UUUGuUGGcUUuUcGAuAGdTdT AD-55049.1 A-113306.1cuAucGAAAAGcCAAcAAAdTdT A-113302.2 UUUGUUGGCUUUUcGAuAGdTdT AD-55055.1A-113306.2 cuAucGAAAAGcCAAcAAAdTdT A-113303.2 UUUGUUGGCUUuUcGAuAGdTdTAD-55061.1 A-113306.3 cuAucGAAAAGcCAAcAAAdTdT A-113304.2UUUGUUGGcUUuUcGAuAGdTdT AD-55067.1 A-113306.4 cuAucGAAAAGcCAAcAAAdTdTA-113305.2 UUUGuUGGcUUuUcGAuAGdTdT AD-55026.1 A-113307.1cuAucGAAAAGcCAACAAAdTdT A-113302.3 UUUGUUGGCUUUUcGAuAGdTdT AD-55032.1A-113307.2 cuAucGAAAAGcCAACAAAdTdT A-113303.3 UUUGUUGGCUUuUcGAuAGdTdTAD-55038.1 A-113307.3 cuAucGAAAAGcCAACAAAdTdT A-113304.3UUUGUUGGcUUuUcGAuAGdTdT AD-55044.1 A-113307.4 cuAucGAAAAGcCAACAAAdTdTA-113305.3 UUUGuUGGcUUuUcGAuAGdTdT AD-50487.1 A-104637.1ccAuGAAucccAuGuGcAudTsdT A-104638.1 AUGcAcAUGGGAUUcAUGGdTsdT AD-55050.1A-113308.1 ccAuGAAucccAuGuGcAudTdT A-113309.1 AUGcAcAUGGGAUUcAuGGdTdTAD-55056.1 A-113308.2 ccAuGAAucccAuGuGcAudTdT A-113310.1AUGCAcAUGGGAUUcAuGGdTdT AD-55062.1 A-113308.3 ccAuGAAucccAuGuGcAudTdTA-113311.1 AUGCAcAUGGGAuUcAuGGdTdT AD-55068.1 A-113308.4ccAuGAAucccAuGuGcAudTdT A-113312.1 AUGCACAUGGGAuUcAuGgdTdT AD-55027.1A-113313.1 ccAuGAAucccAuGUGcAUdTdT A-113309.2 AUGcAcAUGGGAUUcAuGGdTdTAD-55033.1 A-113313.2 ccAuGAAucccAuGUGcAUdTdT A-113310.2AUGCAcAUGGGAUUcAuGGdTdT AD-55039.1 A-113313.3 ccAuGAAucccAuGUGcAUdTdTA-113311.2 AUGCAcAUGGGAuUcAuGGdTdT AD-55045.1 A-113313.4ccAuGAAucccAuGUGcAUdTdT A-113312.2 AUGCACAUGGGAuUcAuGgdTdT AD-55051.1A-113314.1 ccAuGAAucCcAuGUGcAUdTdT A-113309.3 AUGcAcAUGGGAUUcAuGGdTdTAD-55057.1 A-113314.2 ccAuGAAucCcAuGUGcAUdTdT A-113310.3AUGCAcAUGGGAUUcAuGGdTdT AD-55063.1 A-113314.3 ccAuGAAucCcAuGUGcAUdTdTA-113311.3 AUGCAcAUGGGAuUcAuGGdTdT AD-55069.1 A-113314.4ccAuGAAucCcAuGUGcAUdTdT A-113312.3 AUGCACAUGGGAuUcAuGgdTdT AD-50509.1A-104661.1 cuGccGAcucuAucGAAAAdTsdT A-104662.1 UUUUCGAuAGAGUCGGcAGdTsdTAD-55029.1 A-113321.1 cuGccGAcuCuAuCGAaAAdTdT A-113316.3UUUUCGAuAGAGUCGGcAgdTdT AD-55028.1 A-113315.1 cuGccGAcucuAucGAAAAdTdTA-113316.1 UUUUCGAuAGAGUCGGcAgdTdT AD-55052.1 A-113320.1cuGccGAcuCuAuCGAAAAdTdT A-113316.2 UUUUCGAuAGAGUCGGcAgdTdT AD-55035.1A-113321.2 cuGccGAcuCuAuCGAaAAdTdT A-113317.3 UUUUCGAuAGAGUCgGcAgdTdTAD-55047.1 A-113321.4 cuGccGAcuCuAuCGAaAAdTdT A-113319.3UUuUCGAUAGAGUCgGcAgdTdT AD-55058.1 A-113320.2 cuGccGAcuCuAuCGAAAAdTdTA-113317.2 UUUUCGAuAGAGUCgGcAgdTdT AD-55046.1 A-113315.4cuGccGAcucuAucGAAAAdTdT A-113319.1 UUuUCGAUAGAGUCgGcAgdTdT AD-55070.1A-113320.4 cuGccGAcuCuAuCGAAAAdTdT A-113319.2 UUuUCGAUAGAGUCgGcAgdTdTAD-55034.1 A-113315.2 cuGccGAcucuAucGAAAAdTdT A-113317.1UUUUCGAuAGAGUCgGcAgdTdT AD-55041.1 A-113321.3 cuGccGAcuCuAuCGAaAAdTdTA-113318.3 UUuUCGAuAGAGUCgGcAgdTdT AD-55064.1 A-113320.3cuGccGAcuCuAuCGAAAAdTdT A-113318.2 UUuUCGAuAGAGUCgGcAgdTdT AD-55040.1A-113315.3 cuGccGAcucuAucGAAAAdTdT A-113318.1 UUuUCGAuAGAGUCgGcAgdTdTAD-50534.1 A-104769.1 ucuucuuuGccAAAcuGAAdTsdT A-104770.1UUcAGUUUGGcAAAGAAGAdTsdT AD-55053.1 A-113322.1 ucuucuuuGccAAAcuGAAdTdTA-113323.1 UUcAGUUUGGcAAAGAAGadTdT AD-55030.1 A-113327.1ucuucuuuGcCAAACuGAAdTdT A-113323.2 UUcAGUUUGGcAAAGAAGadTdT AD-55054.1A-113327.5 ucuucuuuGcCAAACuGAAdTdT A-113328.1 UUCAGUUuGGcAAAGAAGadTdTAD-55059.1 A-113322.2 ucuucuuuGccAAAcuGAAdTdT A-113324.1UUcAGUUUGGcAAAgAAGadTdT AD-55036.1 A-113327.2 ucuucuuuGcCAAACuGAAdTdTA-113324.2 UUcAGUUUGGcAAAgAAGadTdT AD-55060.1 A-113327.6ucuucuuuGcCAAACuGAAdTdT A-113329.1 UUCAGUUuGGcAAAgAAGadTdT AD-55071.1A-113322.4 ucuucuuuGccAAAcuGAAdTdT A-113326.1 UUcAGUUUGGCaAAgAAGadTdTAD-55048.1 A-113327.4 ucuucuuuGcCAAACuGAAdTdT A-113326.2UUcAGUUUGGCaAAgAAGadTdT AD-55066.1 A-113327.7 ucuucuuuGcCAAACuGAAdTdTA-113330.1 UUCAGUUuGGcaAAgAAGadTdT AD-55042.1 A-113327.3ucuucuuuGcCAAACuGAAdTdT A-113325.2 UUcAGUUUGGcaAAgAAGadTdT AD-55065.1A-113322.3 ucuucuuuGccAAAcuGAAdTdT A-113325.1 UUcAGUUUGGcaAAgAAGadTdTAD-54944.1 A-113073.1 GfgUfuAfaCfaCfCfAfuUfuA A-113074.1uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg fcUfuCfaAfL96 AD-54951.1 A-112997.1UfaGfuAfuCfaGfCfCfaAfuC A-112998.1 aAfaGfgCfgAfuUfggcUfgAfuAfcUfasAfscfgCfcUfuUfL96 AD-54942.1 A-113041.1 CfgCfuUfuGfcUfAfCfcAfcU A-113042.1aUfaGfaAfaGfuGfguaGfcAfaAfgCfgsGfsg fuUfcUfaUfL96 AD-54948.1 A-113043.1CfuUfuGfcUfaCfCfAfcUfuU A-113044.1 uGfaUfaGfaAfaGfuggUfaGfcAfaAfgsCfsgfcUfaUfcAfL96 AD-54957.1 A-112999.1 AfuCfgAfaAfaGfCfCfaAfcA 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GfGfUfUfAfAfCfaCfCfAfuU A-115880.1uUfgAaGfuAaAfuggUfgUfuAfaCfcsasg fUfAfCfUfUfCfAfAfL96 AD-56372.1A-115868.14 GfGfUfUfAfAfCfaCfCfAfuU A-115881.1uUfgAaguAaAfuggUfgUfuAfaCfcsasg fUfAfCfUfUfCfAfAfL96 AD-56378.1A-115868.15 GfGfUfUfAfAfCfaCfCfAfuU A-115882.1uUfgAaGuAaAfuggUfgUfuAfaCfcsasg fUfAfCfUfUfCfAfAfL96 AD-56384.1A-115868.16 GfGfUfUfAfAfCfaCfCfAfuU A-115883.1uUgAaguAaAfuggUfgUfuAfaCfcsasg fUfAfCfUfUfCfAfAfL96 AD-56390.1A-115868.17 GfGfUfUfAfAfCfaCfCfAfuU A-115884.1uUgAaGuAaAfuggUfgUfuAfaCfcsasg fUfAfCfUfUfCfAfAfL96 AD-56396.1A-113073.7 GfgUfuAfaCfaCfCfAfuUfuA A-115885.1uUfgAfAfGfuAfaAfuggUfgUfUfAfaCfcsAf fcUfuCfaAfL96 sg AD-56402.1A-115863.2 GfgUfUfAfaCfaCfCfAfuUfu A-115885.2uUfgAfAfGfuAfaAfuggUfgUfUfAfaCfcsAf AfcUfuCfaAfL96 sg AD-56408.1A-115864.2 GfgUfUfAfaCfaCfCfAfuUfU A-115885.3uUfgAfAfGfuAfaAfuggUfgUfUfAfaCfcsAf fAfcUfuCfaAfL96 sg AD-56414.1A-115865.2 GfgUfUfAfaCfaCfCfAfuUfU A-115885.4uUfgAfAfGfuAfaAfuggUfgUfUfAfaCfcsAf fAfcUfUfCfaAfL96 sg AD-56373.1A-115886.1 GfgUfUfAfaCfaCfCfAfuUfU A-115885.5uUfgAfAfGfuAfaAfuggUfgUfUfAfaCfcsAf fAfcUfUfCfAfAfL96 sg AD-56379.1A-115866.2 GfgUfUfAfAfCfaCfCfAfuUf A-115885.6uUfgAfAfGfuAfaAfuggUfgUfUfAfaCfcsAf UfAfcUfUfCfAfAfL96 sg AD-56385.1A-115867.2 GfGfUfUfAfAfCfaCfCfAfuU A-115885.7uUfgAfAfGfuAfaAfuggUfgUfUfAfaCfcsAf fUfAfcUfUfCfAfAfL96 sg AD-56391.1A-115868.18 GfGfUfUfAfAfCfaCfCfAfuU A-115885.8uUfgAfAfGfuAfaAfuggUfgUfUfAfaCfcsAf fUfAfCfUfUfCfAfAfL96 sg AD-56397.1A-115869.2 GfGfUfUfAfAfCfAfCfCfAfu A-115885.9uUfgAfAfGfuAfaAfuggUfgUfUfAfaCfcsAf UfUfAfCfUfUfCfAfAfL96 sg AD-56403.1A-113073.8 GfgUfuAfaCfaCfCfAfuUfuA A-115887.1uUfgAfAfGfUfAfaAfuggUfgUfUfAfaCfcsA fcUfuCfaAfL96 fsg AD-56409.1A-115863.3 GfgUfUfAfaCfaCfCfAfuUfu A-115887.2uUfgAfAfGfUfAfaAfuggUfgUfUfAfaCfcsA AfcUfuCfaAfL96 fsg AD-56415.1A-115864.3 GfgUfUfAfaCfaCfCfAfuUfU A-115887.3uUfgAfAfGfUfAfaAfuggUfgUfUfAfaCfcsA fAfcUfuCfaAfL96 fsg AD-56374.1A-115865.3 GfgUfUfAfaCfaCfCfAfuUfU A-115887.4uUfgAfAfGfUfAfaAfuggUfgUfUfAfaCfcsA fAfcUfUfCfaAfL96 fsg AD-56380.1A-115886.2 GfgUfUfAfaCfaCfCfAfuUfU A-115887.5uUfgAfAfGfUfAfaAfuggUfgUfUfAfaCfcsA fAfcUfUfCfAfAfL96 fsg AD-56386.1A-115866.3 GfgUfUfAfAfCfaCfCfAfuUf A-115887.6uUfgAfAfGfUfAfaAfuggUfgUfUfAfaCfcsA UfAfcUfUfCfAfAfL96 fsg AD-56392.1A-115867.3 GfGfUfUfAfAfCfaCfCfAfuU A-115887.7uUfgAfAfGfUfAfaAfuggUfgUfUfAfaCfcsA fUfAfcUfUfCfAfAfL96 fsg AD-56398.1A-115868.19 GfGfUfUfAfAfCfaCfCfAfuU A-115887.8uUfgAfAfGfUfAfaAfuggUfgUfUfAfaCfcsA fUfAfCfUfUfCfAfAfL96 fsg AD-56404.1A-115869.3 GfGfUfUfAfAfCfAfCfCfAfu A-115887.9uUfgAfAfGfUfAfaAfuggUfgUfUfAfaCfcsA UfUfAfCfUfUfCfAfAfL96 fsg AD-56410.1A-115888.1 GfgUfuAfaCfaCfCfAfuUfuA A-113074.19uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg fcUfUfCfAfAfL96 AD-56416.1A-115889.1 GfgUfuAfaCfaCfCfAfuUfuA A-113074.20uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg fCfUfUfCfaAfL96 AD-56375.1A-115890.1 GfgUfuAfaCfaCfCfAfuUfUf A-113074.21uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg AfCfUfuCfaAfL96 AD-56381.1A-115891.1 GfgUfuAfaCfaCfCfAfUfUfU A-113074.22uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg fAfcUfuCfaAfL96 AD-56387.1A-115892.1 GfgUfuAfaCfAfCfCfAfUfUf A-113074.23uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg uAfcUfuCfaAfL96 AD-56393.1A-115893.1 GfgUfuAfAfCfAfCfCfAfuUf A-113074.24uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg uAfcUfuCfaAfL96 AD-56399.1A-115894.1 GfgUfUfAfAfCfaCfCfAfuUf A-113074.25uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg uAfcUfuCfaAfL96 AD-56405.1A-115895.1 GfGfUfUfAfaCfaCfCfAfuUf A-113074.26uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg uAfcUfuCfaAfL96 AD-56411.1A-115896.1 GfgUfuAfaCfaCfCfAfuUfuA A-113074.27uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg fcUfucaaL96 AD-56417.1 A-115897.1GfgUfuAfaCfaCfCfAfuUfuA A-113074.28 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsgfcuucaAfL96 AD-56376.1 A-115898.1 GfgUfuAfaCfaCfCfAfuUfua A-113074.29uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg cuuCfaAfL96 AD-56382.1 A-115899.1GfgUfuAfaCfaCfCfAfuuuac A-113074.30 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsgUfuCfaAfL96 AD-56388.1 A-115900.1 GfgUfuAfaCfaCfcauuuAfcU A-113074.31uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg fuCfaAfL96 AD-56394.1 A-115901.1GfgUfuAfaCfaccauUfuAfcU A-113074.32 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsgfuCfaAfL96 AD-56400.1 A-115902.1 GfgUfuAfacaccAfuUfuAfcU A-113074.33uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg fuCfaAfL96 AD-56406.1 A-115903.1GfgUfuAfacaCfCfAfuuuAfc A-113074.34 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsgUfuCfaAfL96 AD-56412.1 A-115904.1 GfgUfuaacaCfCfAfuUfuAfc A-113074.35uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg UfuCfaAfL96 AD-56418.1 A-115905.1GfguuaaCfaCfCfAfuUfuAfc A-113074.36 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsgUfuCfaAfL96 AD-56377.1 A-115906.1 gguuAfaCfaCfCfAfuUfuAfc A-113074.37uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg UfuCfaAfL96 AD-56383.1 A-113073.9GfgUfuAfaCfaCfCfAfuUfuA A-115907.1 UfUfGfAfaGfuAfaAfuggUfgUfuAfaCfcsAffcUfuCfaAfL96 sg AD-56389.1 A-113073.10 GfgUfuAfaCfaCfCfAfuUfuAA-115908.1 uUfGfAfAfGfuAfaAfuggUfgUfuAfaCfcsAf fcUfuCfaAfL96 sgAD-56395.1 A-113073.11 GfgUfuAfaCfaCfCfAfuUfuA A-115909.1uUfgAfAfGfUfAfaAfuggUfgUfuAfaCfcsAf fcUfuCfaAfL96 sg AD-56401.1A-113073.12 GfgUfuAfaCfaCfCfAfuUfuA A-115910.1uUfgAfaGfUfAfAfAfuggUfgUfuAfaCfcsAf fcUfuCfaAfL96 sg AD-56407.1A-113073.13 GfgUfuAfaCfaCfCfAfuUfuA A-115911.1uUfgAfaGfuAfAfAfUfGfgUfgUfuAfaCfcsA fcUfuCfaAfL96 fsg AD-56413.1A-113073.14 GfgUfuAfaCfaCfCfAfuUfuA A-115912.1uUfgAfaGfuAfaAfUfGfGfUfgUfuAfaCfcsA fcUfuCfaAfL96 fsg AD-56419.2A-113073.15 GfgUfuAfaCfaCfCfAfuUfuA A-115913.1uUfgAfaGfuAfaAfuGfGfUfGfUfuAfaCfcsA fcUfuCfaAfL96 fsg AD-56425.2A-113073.16 GfgUfuAfaCfaCfCfAfuUfuA A-115914.1uUfgAfaGfuAfAfAfuggUfGfUfuAfaCfcsAf fcUfuCfaAfL96 sg AD-56431.2A-113073.17 GfgUfuAfaCfaCfCfAfuUfuA A-115915.1uUfgAfaGfuAfaAfuggUfGfUfUfAfaCfcsAf fcUfuCfaAfL96 sg AD-56436.2A-113073.18 GfgUfuAfaCfaCfCfAfuUfuA A-115916.1uUfgAfaGfuAfaAfuggUfgUfUfAfAfCfcsAf fcUfuCfaAfL96 sg AD-56442.2A-113073.19 GfgUfuAfaCfaCfCfAfuUfuA A-115917.1uUfgAfaGfuAfaAfuggUfgUfuAfAfCfCfsAf fcUfuCfaAfL96 sg AD-56448.2A-113073.20 GfgUfuAfaCfaCfCfAfuUfuA A-115918.1uUfgAfaGfuAfaAfuggUfgUfuAfaCfCfsAfs fcUfuCfaAfL96 Gf AD-56454.2A-113073.21 GfgUfuAfaCfaCfCfAfuUfuA A-115919.1uugaaGfuAfaAfuggUfgUfuAfaCfcsAfsg fcUfuCfaAfL96 AD-56460.2 A-113073.22GfgUfuAfaCfaCfCfAfuUfuA A-115920.1 uUfgaaguAfaAfuggUfgUfuAfaCfcsAfsgfcUfuCfaAfL96 AD-56420.2 A-113073.23 GfgUfuAfaCfaCfCfAfuUfuA A-115921.1uUfgAfaguaaAfuggUfgUfuAfaCfcsAfsg fcUfuCfaAfL96 AD-56426.2 A-113073.24GfgUfuAfaCfaCfCfAfuUfuA A-115922.1 uUfgAfaGfuaaauggUfgUfuAfaCfcsAfsgfcUfuCfaAfL96 AD-56432.2 A-113073.25 GfgUfuAfaCfaCfCfAfuUfuA A-115923.1uUfgAfaGfuAfaauggugUfuAfaCfcsAfsg fcUfuCfaAfL96 AD-56437.2 A-113073.26GfgUfuAfaCfaCfCfAfuUfuA A-115924.1 uUfgAfaGfuAfaAfugguguuAfaCfcsAfsgfcUfuCfaAfL96 AD-56443.2 A-113073.27 GfgUfuAfaCfaCfCfAfuUfuA A-115925.1uUfgAfaGfuAfaAfuggUfguuaaCfcsAfsg fcUfuCfaAfL96 AD-56449.2 A-113073.28GfgUfuAfaCfaCfCfAfuUfuA A-115926.1 uUfgAfaGfuAfaAfuggUfgUfuaaccsAfsgfcUfuCfaAfL96 AD-56455.2 A-113073.29 GfgUfuAfaCfaCfCfAfuUfuA A-115927.1uUfgAfaGfuAfaAfuggUfgUfuAfaccsasg fcUfuCfaAfL96 AD-56461.2 A-115888.2GfgUfuAfaCfaCfCfAfuUfuA A-115919.2 uugaaGfuAfaAfuggUfgUfuAfaCfcsAfsgfcUfUfCfAfAfL96 AD-56421.2 A-115889.2 GfgUfuAfaCfaCfCfAfuUfuA A-115920.2uUfgaaguAfaAfuggUfgUfuAfaCfcsAfsg fCfUfUfCfaAfL96 AD-56427.2 A-115890.2GfgUfuAfaCfaCfCfAfuUfUf A-115921.2 uUfgAfaguaaAfuggUfgUfuAfaCfcsAfsgAfCfUfuCfaAfL96 AD-56433.2 A-115891.2 GfgUfuAfaCfaCfCfAfUfUfU A-115922.2uUfgAfaGfuaaauggUfgUfuAfaCfcsAfsg fAfcUfuCfaAfL96 AD-56438.2 A-115892.2GfgUfuAfaCfAfCfCfAfUfUf A-115923.2 uUfgAfaGfuAfaauggugUfuAfaCfcsAfsguAfcUfuCfaAfL96 AD-56444.2 A-115893.2 GfgUfuAfAfCfAfCfCfAfuUf A-115924.2uUfgAfaGfuAfaAfugguguuAfaCfcsAfsg uAfcUfuCfaAfL96 AD-56450.2 A-115894.2GfgUfUfAfAfCfaCfCfAfuUf A-115925.2 uUfgAfaGfuAfaAfuggUfguuaaCfcsAfsguAfcUfuCfaAfL96 AD-56456.2 A-115895.2 GfGfUfUfAfaCfaCfCfAfuUf A-115926.2uUfgAfaGfuAfaAfuggUfgUfuaaccsAfsg uAfcUfuCfaAfL96 AD-56462.2 A-115896.2GfgUfuAfaCfaCfCfAfuUfuA A-115907.2 UfUfGfAfaGfuAfaAfuggUfgUfuAfaCfcsAffcUfucaaL96 sg AD-56422.2 A-115897.2 GfgUfuAfaCfaCfCfAfuUfuA A-115908.2uUfGfAfAfGfuAfaAfuggUfgUfuAfaCfcsAf fcuucaAfL96 sg AD-56428.2 A-115898.2GfgUfuAfaCfaCfCfAfuUfua A-115909.2 uUfgAfAfGfUfAfaAfuggUfgUfuAfaCfcsAfcuuCfaAfL96 sg AD-56434.2 A-115899.2 GfgUfuAfaCfaCfCfAfuuuac A-115910.2uUfgAfaGfUfAfAfAfuggUfgUfuAfaCfcsAf UfuCfaAfL96 sg AD-56439.2 A-115900.2GfgUfuAfaCfaCfcauuuAfcU A-115911.2 uUfgAfaGfuAfAfAfUfGfgUfgUfuAfaCfcsAfuCfaAfL96 fsg AD-56445.2 A-115901.2 GfgUfuAfaCfaccauUfuAfcU A-115912.2uUfgAfaGfuAfaAfUfGfGfUfgUfuAfaCfcsA fuCfaAfL96 fsg AD-56451.2 A-115902.2GfgUfuAfacaccAfuUfuAfcU A-115913.2 uUfgAfaGfuAfaAfuGfGfUfGfUfuAfaCfcsAfuCfaAfL96 fsg AD-56457.2 A-115903.2 GfgUfuAfacaCfCfAfuuuAfc A-115914.2uUfgAfaGfuAfAfAfuggUfGfUfuAfaCfcsAf UfuCfaAfL96 sg AD-56463.2 A-115904.2GfgUfuaacaCfCfAfuUfuAfc A-115915.2 uUfgAfaGfuAfaAfuggUfGfUfUfAfaCfcsAfUfuCfaAfL96 sg AD-56423.2 A-115905.2 GfguuaaCfaCfCfAfuUfuAfc A-115916.2uUfgAfaGfuAfaAfuggUfgUfUfAfAfCfcsAf UfuCfaAfL96 sg AD-56429.2 A-115906.2gguuAfaCfaCfCfAfuUfuAfc A-115917.2 uUfgAfaGfuAfaAfuggUfgUfuAfAfCfCfsAfUfuCfaAfL96 sg AD-56440.2 A-113073.31 GfgUfuAfaCfaCfCfAfuUfuA A-115928.1uUfgAfaGfuAfaAfugGfUfgUfuAfaCfcsAfs fcUfuCfaAfL96 g AD-56446.2A-115929.1 GfgUfuAfaCfacCfAfuUfuAf A-115928.2uUfgAfaGfuAfaAfugGfUfgUfuAfaCfcsAfs cUfuCfaAfL96 g AD-56452.2A-113073.32 GfgUfuAfaCfaCfCfAfuUfuA A-115930.1UUfgAfaGfuAfaAfugGfUfgUfuAfaCfcsAfs fcUfuCfaAfL96 g AD-56458.2A-115929.2 GfgUfuAfaCfacCfAfuUfuAf A-115930.2UUfgAfaGfuAfaAfugGfUfgUfuAfaCfcsAfs cUfuCfaAfL96 g AD-56464.2A-113073.33 GfgUfuAfaCfaCfCfAfuUfuA A-115931.1uUfgAfaGfuAfaAfugGUfgUfuAfaCfcsAfsg fcUfuCfaAfL96 AD-56424.2 A-115929.3GfgUfuAfaCfacCfAfuUfuAf A-115931.2 uUfgAfaGfuAfaAfugGUfgUfuAfaCfcsAfsgcUfuCfaAfL96 AD-56430.2 A-115932.1 GfgUfuAfaCfaCCfAfuUfuAf A-115931.3uUfgAfaGfuAfaAfugGUfgUfuAfaCfcsAfsg cUfuCfaAfL96 AD-56435.2 A-113073.34GfgUfuAfaCfaCfCfAfuUfuA A-115933.1 UUfgAfaGfuAfaAfugGUfgUfuAfaCfcsAfsgfcUfuCfaAfL96 AD-56441.2 A-115929.4 GfgUfuAfaCfacCfAfuUfuAf A-115933.2UUfgAfaGfuAfaAfugGUfgUfuAfaCfcsAfsg cUfuCfaAfL96 AD-56447.2 A-115932.2GfgUfuAfaCfaCCfAfuUfuAf A-115933.3 UUfgAfaGfuAfaAfugGUfgUfuAfaCfcsAfsgcUfuCfaAfL96 AD-56453.2 A-115934.1 GfgUfuAfaCfaUfCfAfuUfuA A-113074.39uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg fcUfuCfaAfL96 AD-56459.2 A-115935.1GfgUfuAfaCfaAfCfAfuUfuA A-113074.40 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsgfcUfuCfaAfL96 AD-56465.2 A-115936.1 GfgUfuAfaCfaCfUfAfuUfuA A-113074.41uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg fcUfuCfaAfL96 AD-56471.2 A-115937.1GfgUfuAfaCfaCfAfAfuUfuA A-113074.42 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsgfcUfuCfaAfL96 AD-56477.2 A-113073.35 GfgUfuAfaCfaCfCfAfuUfuA A-115938.1uUfgAfaGfuAfaAfuggUfgUfuAfaCfc fcUfuCfaAfL96 AD-56483.3 A-113073.36GfgUfuAfaCfaCfCfAfuUfuA A-115939.1 uUfgAfaGfuAfaAfuggUfgUfuAfaCfscfcUfuCfaAfL96 AD-56488.2 A-113073.37 GfgUfuAfaCfaCfCfAfuUfuA A-115940.1uUfgAfaGfuAfaAfuggUfgUfuAfasCfsc fcUfuCfaAfL96 AD-56483.4 A-113073.38GfgUfuAfaCfaCfCfAfuUfuA A-115939.2 uUfgAfaGfuAfaAfuggUfgUfuAfaCfscfcUfuCfaAfL96 AD-56497.2 A-115941.1 UfuAfaCfaCfCfAfuUfuAfcU A-115940.2uUfgAfaGfuAfaAfuggUfgUfuAfasCfsc fuCfaAfL96 AD-56502.2 A-115941.2UfuAfaCfaCfCfAfuUfuAfcU A-115942.1 uUfgAfaGfuAfaAfuggUfgUfuAfafuCfaAfL96 AD-56466.2 A-115941.3 UfuAfaCfaCfCfAfuUfuAfcU A-115943.1uUfgAfaGfuAfaAfuggUfgUfuAfsa fuCfaAfL96 AD-56472.2 A-115941.4UfuAfaCfaCfCfAfuUfuAfcU A-115944.1 uUfgAfaGfuAfaAfuggUfgUfusAfsafuCfaAfL96 AD-56478.2 A-115945.1 AfaCfaCfCfAfuUfuAfcUfuC A-115943.2uUfgAfaGfuAfaAfuggUfgUfuAfsa faAfL96 AD-56484.2 A-115945.2AfaCfaCfCfAfuUfuAfcUfuC A-115944.2 uUfgAfaGfuAfaAfuggUfgUfusAfsa faAfL96AD-56493.2 A-113073.39 GfgUfuAfaCfaCfCfAfuUfuA A-115948.1 uUfgAf(Aeo)fcUfuCfaAfL96 GfuAfaAfuggUfgUfuAfaCfcsAfsg AD-56498.2 A-113073.40GfgUfuAfaCfaCfCfAfuUfuA A-115949.1 uUfgAfa(Geo) fcUfuCfaAfL96uAfaAfuggUfgUfuAfaCfcsAfsg AD-56467.2 A-113073.42GfgUfuAfaCfaCfCfAfuUfuA A-115951.1 uUfgAfaGfu(Aeo) fcUfuCfaAfL96aAfuggUfgUfuAfaCfcsAfsg AD-56473.2 A-113073.43 GfgUfuAfaCfaCfCfAfuUfuAA-115952.1 uUfgAfaGfuAf(Aeo) fcUfuCfaAfL96 AfuggUfgUfuAfaCfcsAfsgAD-56479.2 A-113073.44 GfgUfuAfaCfaCfCfAfuUfuA A-115953.1uUfgAfaGfuAfa(Aeo) fcUfuCfaAfL96 uggUfgUfuAfaCfcsAfsg AD-56485.2A-113073.45 GfgUfuAfaCfaCfCfAfuUfuA A-115954.1 uUfgAfaGfuAfaAfugg(Teo)fcUfuCfaAfL96 gUfuAfaCfcsAfsg AD-56490.2 A-113073.46GfgUfuAfaCfaCfCfAfuUfuA A-115955.1 uUfgAfaGfuAfaAfuggUf(Geo)fcUfuCfaAfL96 UfuAfaCfcsAfsg AD-56494.2 A-113073.47GfgUfuAfaCfaCfCfAfuUfuA A-115956.1 uUfgAfaGfuAfaAfuggUfg(Teo)fcUfuCfaAfL96 uAfaCfcsAfsg AD-56499.2 A-113073.48GfgUfuAfaCfaCfCfAfuUfuA A-115957.1 uUfgAfaGfuAfaAfuggUfgUf(Teo)fcUfuCfaAfL96 AfaCfcsAfsg AD-56504.2 A-113073.49 GfgUfuAfaCfaCfCfAfuUfuAA-115958.1 uUfgAfaGfuAfaAfuggUfgUfu(Aeo) fcUfuCfaAfL96 aCfcsAfsgAD-56468.2 A-113073.50 GfgUfuAfaCfaCfCfAfuUfuA A-115959.1uUfgAfaGfuAfaAfuggUfgUfuAf(Aeo) fcUfuCfaAfL96 CfcsAfsg AD-56474.2A-113073.51 GfgUfuAfaCfaCfCfAfuUfuA A-115960.1uUfgAfaGfuAfaAfuggUfgUfuAfa(m5Ceo) fcUfuCfaAfL96 csAfsg AD-56480.2A-113073.52 GfgUfuAfaCfaCfCfAfuUfuA A-115961.1uUfgAfaGfuAfaAfuggUfgUfuAfaCf fcUfuCfaAfL96 (m5Ceos)Afsg AD-56486.2A-113073.53 GfgUfuAfaCfaCfCfAfuUfuA A-115962.1uUfgAfaGfuAfaAfuggUfgUfuAfaCfcs fcUfuCfaAfL96 (Aeos)(Geo) AD-56491.2A-113073.54 GfgUfuAfaCfaCfCfAfuUfuA A-115963.1uUfgAfaGfuAfaAf(Teo)(Geo)(Geo)UfgUf fcUfuCfaAfL96 usAfsa AD-56495.2A-115964.1 GfgUfuAfaCfaCfCfAfuUfuA A-113074.43uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg fcUf(Teo)Cf(Aeo)AfL96 AD-56500.2A-115965.1 Gf(Geo)UfuAfaCfaCfCfAfu A-113074.44uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg UfuAfcUf(Teo)Cf(Aeo)AfL 96AD-56505.2 A-115966.1 Gf(Geo)Uf(Teo)AfaCfaCfC A-113074.45uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg fAfuUfuAfcUf(Teo)Cf (Aeo)AfL96AD-56469.2 A-115967.1 Gf(Geo)Uf(Teo)AfaCfaCfC A-113074.46uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg fAfuUfuAf(m5Ceo)Uf(Teo) Cf(Aeo)AfL96AD-56475.2 A-115968.1 Gf(Geo)Uf(Teo)AfaCfaCfC A-113074.47uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg fAfuUf(Teo)Af(m5Ceo)Uf(Teo)Cf(Aeo)AfL96 AD-56481.2 A-115969.1 Gf(Geo)Uf(Teo)AfaCfaCfCA-113074.48 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg fAf(Teo)Uf(Teo)Af(m5Ceo)Uf(Teo)Cf(Aeo)Af L96 AD-56487.2 A-115970.1Gf(Geo)Uf(Teo)Af(Aeo)Cf A-113074.49 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg(Aeo)CfCfAf(Teo)Uf(Teo) Af(m5Ceo)Uf(Teo)Cf(Aeo) AfL96

Example 3 LNP-Mediated Delivery of siRNAs

Based on the in vitro single dose and IC₅₀ results described above,modified AD-50509 was selected for formulation in a lipid nanoparticle(LNP). In order to determine an effective dose for LNP-mediated deliveryof AD-50509, CD1 mice were intravenously injected with a single dose ofan LNP formulation (AF-011) of AD-50509 siRNA at 0.003, 0.01, 0.03, 0.1,0.3, or 1.0 mg/kg. Animals were sacrificed 48 hours later and the levelof Serpinc1 mRNA relative to GAPDH and the level of Serpinc1 proteinwere determined as described herein. As shown in FIGS. 3A and 3B, themaximum Serpinc1 mRNA silencing of 85% with AF-011-AD-50509 was achievedwith an ED₅₀ of about 0.1 mg/kg (FIG. 3A) and the maximum Serpinc1protein silencing of 90% was achieved with an ED₅₀ of about 0.05 mg/kg(FIG. 3B).

The duration of silencing of an LNP formulation of AD-50509 siRNA(AF-011-50509) was determined in CD1 mice following a single 1 mg/kgintravenous injection of the siRNA. Animals were sacrificed at Day 1, 2,3, 7, 14, 21, or 28 after administration and the relative level ofSerpinc1 mRNA and the level of Serpinc1 protein were determined. FIG. 4Ademonstrates that AF-011 formulated AD-50509 achieved Serpinc1 mRNAsilencing of about 90% within 24 hours of administration and that therewas approximately a 50% recovery in the relative amount of Serpinc1 mRNAby about two weeks after administration. FIG. 4B demonstrates thatAF-011 formulated AD-50509 achieved Serpinc1 protein silencing of about90% within about 72 hours of administration and that there wasapproximately a 50% recovery in the relative amount of Serpinc1 proteinby about two weeks after administration. Serpinc1 activity was alsodetermined by measuring Factor Xa activity using a commerciallyavailable kit (Aniara) in CD1 mice following a single 1 mg/kgintravenous injection of the LNP formulated AD-50509 siRNA. Animals weresacrificed at Day 1, 2, 3, 7, 14, 21, or 28 after administration and therelative activity level of Serpinc1 protein and the relative Serpinc1protein level were determined. FIG. 4C shows that there is goodcorrelation between the level of Serpinc1 protein level and Serpinc1activity.

Example 4 GalNAc-Conjugated siRNAs

Forty-four modified Serpinc1 siRNA duplexes were conjugated with atrivalent GALNAc at the 3-end of the sense strand. These duplexes wereassayed for efficacy in single dose free uptake of the conjugatedduplexes in Cynomolgus monkey hepatocytes. Table 9 shows the results ofthese assays.

TABLE 9 GalNAc Free-Uptake Single Dose Duplex ID Ave 100 nM Ave 10 nMAve 0.1 nM AD-54944.1 0.4 0.53 0.94 AD-54951.1 0.37 0.56 1 AD-54942.10.38 0.58 1.01 AD-54948.1 0.36 0.6 0.96 AD-54957.1 0.47 0.61 1AD-54933.1 0.51 0.65 0.96 AD-54962.1 0.48 0.66 0.95 AD-54972.1 0.49 0.661.05 AD-54949.1 0.49 0.71 0.96 AD-54936.1 0.54 0.72 1.07 AD-54971.1 0.490.72 1 AD-54955.1 0.52 0.74 0.98 AD-54953.1 0.63 0.76 1.07 AD-54937.10.64 0.81 0.94 AD-54967.1 0.74 0.82 1.02 AD-54935.1 0.68 0.83 0.99AD-54976.1 0.7 0.85 1.04 AD-54965.1 0.7 0.86 0.97 AD-54959.1 0.79 0.860.95 AD-54943.1 0.75 0.86 0.94 AD-54956.1 0.86 0.87 0.95 AD-54973.1 0.960.89 1 AD-54975.1 0.67 0.89 0.99 AD-54963.1 0.73 0.9 0.96 AD-54978.10.85 0.9 0.98 AD-54952.1 0.59 0.91 1.11 AD-54950.1 0.89 0.91 0.95AD-54964.1 0.87 0.93 1.01 AD-54974.1 0.83 0.93 0.96 AD-54969.1 0.87 0.940.94 AD-54961.1 0.74 0.94 1.07 AD-54968.1 0.89 0.95 0.91 AD-54947.1 0.920.96 0.94 AD-54941.1 0.91 0.96 1 AD-54966.1 0.93 0.97 1.06 AD-54940.10.86 0.99 1.03 AD-54958.1 0.97 0.99 1.06 AD-54938.1 0.93 0.99 1.05AD-54934.1 0.92 1 0.96 AD-54939.1 0.84 1.02 1.02 AD-54960.1 0.98 1.031.02 AD-54954.1 1.04 1.03 1.01 AD-54970.1 1.03 1.06 1.01 AD-54946.1 0.831.17 1.1

These duplexes were also assayed for dose response in free uptake andtransfection assays.

Table 10 shows the results of these assays and the rank order of theduplexes for both free uptake and transfection. The 5 duplexes with thebest IC₅₀ are shaded in light gray and the bottom 5 duplexes are shadedin dark gray. The IC₅₀ rank order of the duplexes is well conservedbetween free uptake and transfection-mediated uptake of GalNAcconjugates.

TABLE 10 Dose Response of GalNAc-conjugated duplexes: Free uptake andTransfection

Example 5 AD-54944 Optimization

As described in Example 4 above, AD-54944 was among the most activeGalNAc-conjugated siRNA duplex as determined by both free uptake andtransfection assays and was, thus, selected for further optimization andin vivo testing.

Twenty-nine compounds were prepared based on the same AD-54944 parentsequence and screened for in vivo efficacy using a single 10 mg/kg dose.Animals (C57BL/6) were injected subcutaneously at Day 0 and sacrificedat Day 3. Serum Serpinc1 protein levels were determined by ELISA assayand the level of Serpinc1 mRNA was determined by QRT-PCR using liversamples from the animals. Tables 11 and 12 show the sequences of theduplexes and the results of the single dose screen with these duplexesas a percent knock-down of Serpinc1 protein levels from PBS. FIG. 5shows the results of the single dose screen as a percent knock-down ofSerpince mRNA and protein levels from PBS.

TABLE 11 AD-54944 optimized sequences and protein levels.(The “Sense Sequence” column sequences are disclosed asSEQ ID NOS 779-808, respectively, in order of appearance,and the “Antisense Sequence” column sequences aredisclosed as SEQ ID NOS 809-838, respectively, in order of appearance)Duplex  % Std Name Sense Sequence Antisense Sequence PBS Dev AD-54944GfgUfuAfaCfaCfCfAfuUfuAfcUfu uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsA 66.8 4.8(new) CfaAfL96 fsg AD-56345 GfgUfuAfaCfaCfCfAfuUfuAfcUfuuUfgaaGfuAfaAfuggUfguuAfaCfcsasg 83.6 19.2 CfaAfL96 AD-56351GfguuAfaCfaCfCfAfuUfuAfcUfuC uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 71.7 7.5faAfL96 AD-56363 GfguuAfaCfaCfCfAfuUfuacUfuCfuUfgaaGfuAfaAfuggUfguuAfaCfcsasg 57.9 7.6 aaL96 AD-56334GfguuAfacaCfCfAfuuuacUfucaaL uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 59.4 6.096 AD-56346 GfguuaacaCfCfAfuuuacuucaaL96uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 82.3 13.2 AD-56352GfgUfUfAfaCfaCfCfAfuUfuAfcUf uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsA 52.2 3.6uCfaAfL96 fsg AD-56410 GfgUfuAfaCfaCfCfAfuUfuAfcUfUuUfgAfaGfuAfaAfuggUfgUfuAfaCfcsA 55.0 2.5 fCfAfAfL96 fsg AD-56405GfGfUfUfAfaCfaCfCfAfuUfuAfcU uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsA 60.6 3.1fuCfaAfL96 fsg AD-56383 GfgUfuAfaCfaCfCfAfuUfuAfcUfuUfUfGfAfaGfuAfaAfuggUfgUfuAfaCfc 66.7 7.6 CfaAfL96 sAfsg AD-56730GfgUfuAfaCfaCfCfAfuUfuAfcUfU uUfgAfaGfuAfaAfuggUfgUfuAfaCfCfs 64.4 3.4fCfAfAfL96 AfsGf AD-56380 GfgUfUfAfaCfaCfCfAfuUfUfAfcUuUfgAfAfGfUfAfaAfuggUfgUfUfAfaCf 75.7 5.0 fUfCfAfAfL96 csAfsg AD-56397GfGfUfUfAfAfCfAfCfCfAfuUfUfA uUfgAfAfGfuAfaAfuggUfgUfUfAfaCfc 76.9 0.8fCfUfUfCfAfAfL96 sAfsg AD-56434 GfgUfuAfaCfaCfCfAfuuuacUfuCfuUfgAfaGfUfAfAfAfuggUfgUfuAfaCfc 52.0 5.1 aAfL96 sAfsg AD-56488GfgUfuAfaCfaCfCfAfuUfuAfcUfu uUfgAfaGfuAfaAfuggUfgUfuAfasCfsc 85.3 16.6CfaAfL96 AD-56486 GfgUfuAfaCfaCfCfAfuUfuAfcUfuuUfgAfaGfuAfaAfuggUfgUfuAfaCfcs 67.7 0.5 CfaAfL96 (Aeos)(Geo) AD-56505Gf(Geo)Uf(Teo)AfaCfaCfCfAfuU uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsA 59.7 0.2fuAfcUf(Teo)Cf(Aeo)AfL96 fsg AD-56736 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUuUfgAfaGfuAfaAfuggUf(Geo)UfuAfaC 49.7 1.2 f(Teo)Af(m5Ceo)Uf(Teo)CffcsAfsg (Aeo)AfL96 AD-56738 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUuUfgaaGfuAfaAfuggUfguuAfaCfcsasg 65.0 3.6 fuAfcUf(Teo)Cf(Aeo)AfL96AD-56739 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuU uUfgaaGfuAfaAfuggUfguuAfaCfcsasg57.6 2.1 f(Teo)Af(m5Ceo)Uf(Teo)Cf (Aeo)AfL96 AD-56740Gf(Geo)Uf(Teo)AfaCfaCfCfAfuU uUfgaaGfuAfaAfuggUfgUfuAfaCfcsas 85.5 14.8fuAfcUf(Teo)Cf(Aeo)AfL96 g AD-56743 Gf(Geo)UfuAfaCfaCfCfAfuUfuAfuUfgaaGfuAfaAfuggUfgUfuAfaCfcsas 48.3 3.9 cUf(Teo)Cf(Aeo)AfL96 gAD-56745 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuU uUfgaaGfuAfaAfuggUfgUfuAfaCfcsas54.4 9.9 fuAf(m5Ceo)Uf(Teo)Cf(Aeo)AfL g 96 AD-56454GfgUfuAfaCfaCfCfAfuUfuAfcUfu uugaaGfuAfaAfuggUfgUfuAfaCfcsAfs 72.0 11.0CfaAfL96 g AD-56449 GfgUfuAfaCfaCfCfAfuUfuAfcUfuuUfgAfaGfuAfaAfuggUfgUfuaaccsAfs 54.4 15.9 CfaAfL96 g AD-56746Gf(Geo)UfuAfaCfaCfCfAfuUfuAf uUfgAfaGfuAfaAfuggUfgUfuaaccsAfs 64.7 17.2cUf(Teo)Cf(Aeo)AfL96 g AD-56333 GfguuAfacaCfCfAfuuuacuucaaLuUfgaaGfuAfaAfuggUfgUfuAfaCfcsas 71.5 11.3 96 g AD-56382GfgUfuAfaCfaCfCfAfuuuacUfuCf uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsA 65.7 2.7aAfL96 fsg AD-56748 GfgUfuAfaCfaCfCfAfuuuacUfuCfuUfgaaGfuAfaAfuggUfguuAfaCfcsasg 99.4 19.1 aAfL96 AD-54944GfgUfuAfaCfaCfCfAfuUfuAfcUfu uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsA 46.9 1.7(original) CfaAfL96 fsg control 96.0 8.5 PBS 100.0 4.7

TABLE 12 AD-54944 optimized sequences and protein levels.(The “Sense Sequence” column sequences are disclosed asSEQ ID NOS 839-868, respectively, in order of appearance,and the “Antisense Sequence” column sequences are disclosedas SEQ ID NOS 869-898, respectively, in order of appearance) Duplex %Std Name Sense Sequence Antisense Sequence PBS Dev AD-54944GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg46.9 1.7 (original) AD-56743 Gf(Geo)UfuAfaCfaCfCfAfuUfuAfcUf(Teo)uUfgaaGfuAfaAfuggUfgUfuAfaCfcsasg 48.3 3.9 Cf(Aeo)AfL96 AD-56736Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUf(Teo)AfuUfgAfaGfuAfaAfuggUf(Geo)UfuAfaCfcsAfsg 49.7 1.2(m5Ceo)Uf(Teo)Cf(Aeo)AfL96 AD-56434 GfgUfuAfaCfaCfCfAfuuuacUfuCfaAfL96uUfgAfaGfUfAfAfAfuggUfgUfuAfaCfcsAfsg 52.0 5.1 AD-56352GfgUfUfAfaCfaCfCfAfuUfuAfcUfuCfaAfL96uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 52.2 3.6 AD-56745Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAf(m5Ceo)uUfgaaGfuAfaAfuggUfgUfuAfaCfcsasg 54.4 9.9 Uf(Teo)Cf(Aeo)AfL96 AD-56449GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuaaccsAfsg54.4 15.9 AD-56410 GfgUfuAfaCfaCfCfAfuUfuAfcUfUfCfAfAfL96uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 55.0 2.5 AD-56739Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUf(Teo)Af uUfgaaGfuAfaAfuggUfguuAfaCfcsasg57.6 2.1 (m5Ceo)Uf(Teo)Cf(Aeo)AfL96 AD-56363GfguuAfaCfaCfCfAfuUfuacUfuCfaaL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 57.97.6 AD-56334 GfguuAfacaCfCfAfuuuacUfucaaL96uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 59.4 6.0 AD-56505Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAfcUf uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg59.7 0.2 (Teo)Cf(Aeo)AfL96 AD-56405GfGfUfUfAfaCfaCfCfAfuUfuAfcUfuCfaAfL96uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 60.6 3.1 AD-56730GfgUfuAfaCfaCfCfAfuUfuAfcUfUfCfAfAfL96uUfgAfaGfuAfaAfuggUfgUfuAfaCfCfsAfsGf 64.4 3.4 AD-56746Gf(Geo)UfuAfaCfaCfCfAfuUfuAfcUf(Teo)Cf uUfgAfaGfuAfaAfuggUfgUfuaaccsAfsg64.7 17.2 (Aeo)AfL96 AD-56738 Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAfcUfuUfgaaGfuAfaAfuggUfguuAfaCfcsasg 65.0 3.6 (Teo)Cf(Aeo)AfL96 AD-56382GfgUfuAfaCfaCfCfAfuuuacUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg65.7 2.7 AD-56383 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96UfUfGfAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg 66.7 7.6 AD-54944GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgAfaGfuAfaAfuggUfgUfuAfaCfcsAfsg66.8 4.8 (new) AD-56486 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96uUfgAfaGfuAfaAfuggUfgUfuAfaCfcs 67.7 0.5 (Aeos)(Geo) AD-56333GfguuAfacaCfCfAfuuuacuucaaL96 uUfgaaGfuAfaAfuggUfgUfuAfaCfcsasg 71.511.3 AD-56351 GfguuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 71.7 7.5 AD-56454GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uugaaGfuAfaAfuggUfgUfuAfaCfcsAfsg72.0 11.0 AD-56380 GfgUfUfAfaCfaCfCfAfuUfUfAfcUfUfCuUfgAfAfGfUfAfaAfuggUfgUfUfAfaCfcsAfsg 75.7 5.0 fAfAfL96 AD-56397GfGfUfUfAfAfCfAfCfCfAfuUfUfAfCfU uUfgAfAfGfuAfaAfuggUfgUfUfAfaCfcsAfsg76.9 0.8 fUfCfAfAfL96 AD-56346 GfguuaacaCfCfAfuuuacuucaaL96uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 82.3 13.2 AD-56345GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg83.6 19.2 AD-56488 GfgUfuAfaCfaCfCfAfuUfuAfcUfuCfaAfL96uUfgAfaGfuAfaAfuggUfgUfuAfasCfsc 85.3 16.6 AD-56740Gf(Geo)Uf(Teo)AfaCfaCfCfAfuUfuAfcUf uUfgaaGfuAfaAfuggUfgUfuAfaCfcsasg85.5 14.8 (Teo)Cf(Aeo)AfL96 96.0 8.5 AD-56748GfgUfuAfaCfaCfCfAfuuuacUfuCfaAfL96 uUfgaaGfuAfaAfuggUfguuAfaCfcsasg 99.419.1 PBS 100.0 4.7

The in vivo dose response of AD-54944 conjugated to GalNAc wasdetermined by administering a single subcutaneous dose to C57BL/6J mice(n=5). AD-54944 conjugated to GalNAc was also administeredsubcutaneously as a repeat daily dose of 5 mg/kg to C57BL/6J mice (n=5)over a 5 day period. Animals were sacrificed 72 hours afteradministration and Serpinc1 protein and activity levels were determinedin liver and serum samples as described above.

As shown in FIG. 6, a single subcutaneous dose of AD-54944 conjugated toGalNAc resulted in a protein EC₅₀ of about 10 mg/kg and a 5×5 mg/kgdaily, repeat dose resulted in about a 75% protein silencing.

Additional repeat-dosing of AD-54944 conjugated to GalNAc in C57BL/6Jmice was also performed over an 8 week period to determine the efficacyand duration of silencing. FIGS. 7A and 7B show the results of thesestudies.

Example 6 Dose Duration of a Split-Dose of AD-54944

In order to further evaluate compound AD-54944 knock-down of Serpinc1expression and activity, a split-dosing experiment was performed.C57BL/6 mice were subcutaneously administered GalNAc-conjugated AD-54944and the effect of a 3 times per week, ⅓ dose of AD-54944 was compared tothe effect of a 1 time per week fully concentrated dose of AD-54944. Asummary of the study design is presented in Table 13. Serum Serpinc1protein levels were determined at Days 0, 3, 7, 10, 14, 17, 21, 24, 29,31, and 35.

TABLE 13 Study Design of Split-Dosing Experiment Group Test compoundDose (mg/kg) Frequency 1 AD-54944 1.25 3x/week 2 2.5 (M, W, F) 3 5 4 105 3.75 1x/week 6 7.5 (Monday) 7 15 8 30 9 PBS _(—)

The results of the one-time per week split-dose screen as a percentknock-down of Serpinc1 protein levels from pre-dose levels are shown inFIG. 8 and the results of the three-time per week screen as a percentknock-down of Serpinc1 protein levels from pre-dose levels are shown inFIG. 9. The results demonstrate that there is a dose response effectwith AD-54944 conjugated to GalNAc in both groups and that dosages atboth 30 mg/kg one time per week and at 10 mg/kg three times per weeklead to long-term silencing of Serpinc1.

Example 7 Further Optimization of AD-54944

In order to further improve the efficacy of AD-54944, additionalcompounds were prepared based on the AD-54944 parent sequence. Ingeneral, the modifications included the addition of phosphorothiatelinkages, C16(hexadecyl) modifications, 5′-end-caps, and 2′-methyls. Thenew compounds were screened for in vivo efficacy using both a single 3mg/mg dose and a single 10 mg/kg dose. Animals (C57BL/6) were injectedsubcutaneously at Day 0 and sacrificed at Day 3. Serum Serpinc1 proteinlevels were determined by ELISA assay. The ELISA assay was performedusing an Antithrombin III Mouse ELISA kit purchased from Abcam. Briefly,serum was diluted (e.g., about 1:10,000) and used accordingly tomanufacturers instructions. The plates were read at 450 nm at the end ofthe assay.

Table 14 shows the sequences of the duplexes and the results of thesingle dose screens with these duplexes as a percent knock-down ofSerpinc1 protein levels from PBS. FIGS. 10A and 10B show the results ofthe single dose screen as a percent knock-down of Serpinc1 proteinlevels from PBS. As can be seen in Table 14 and FIG. 10, compoundAD-56813 emerged as a new lead based on the level of knock-down ofSerpinc1 protein levels.

Further compounds were prepared based on the AD-56813 parent sequence inwhich the number of 2′-methoxyethyl and phosphorothioate linkages werereduced in order to determine the minimum chemical modificationsrequired for stability of the compounds which maintained activity of thecompounds. The new compounds were screened for in vivo efficacy usingboth a single 3 mg/mg dose and a single 10 mg/kg dose. Animals (C57BL/6)were injected subcutaneously at Day 0 and sacrificed at Day 3. Serpinc1(AT3) activity and serum Serpinc1 protein levels were determined byELISA assay. The ELISA assay was performed as described above. Serpinc1activity was determined using a BIOPHEN (anti-Factor Xa) activity assaykit. Briefly, serum samples were diluted from about 1:20 to about 1:60and processed according to the manufacturers' instructions. The plateswere read at 450 nm at the end of the assay.

The sequences of the duplexes that were newly prepared and the resultsof the single dose screens with these duplexes as a percent knock-downof Serpinc1 protein levels from PBS are shown in Table 15. FIG. 11 showsthe results of the single dose screen as a percent knock-down ofSerpinc1 protein levels from PBS and FIG. 12 shows the results of thesingle dose screen as a percent knock-down of Serpinc1 activity fromPBS.

As can be seen in Table 15 and FIGS. 11 and 12, although the number ofmodifications to the compound was dramatically reduced, compoundAD-57213 maintained knockdown of Serpinc1 expression and activity and,thus, emerged as a new lead. A single 10 mg/kg dose of AD-57213 led toan ED₉₀ and a single 3 mg/kg dose led to an ED₅₀.

TABLE 14 AD-54944 optimized sequences and protein levels.(The “Sense Sequence” column sequences are disclosed asSEQ ID NOS 899-919, respectively, in order of appearance,and the “Antisense Sequence” column sequences aredisclosed as SEQ ID NOS 920-940, respectively, in order of appearance)3 mpk 10 mpk Sense % % Duplex ID strand Sense sequence AntisenseAntisense sequence PBS stdev PBS stdev AD-56813.2 A-116280.6Gfs(Geo)UfsuAfaCfs A-116278.6 usUfsgAfaGfuAfaAfu 0.62 0.04 0.26 0.04aCfsCfsAfuUfsuAfcU ggUfsgUfsuAfaCfscs fsuCfs(Aeo)AfL96 (Aeos)(Geo)AD-56789.2 A-116276.12 GfgUfsuAfaCfsaCfsC A-116275.7 uUfsgAfaGfuAfaAfug0.74 0.11 0.44 0.11 fsAfuUfsuAfcUfsuCf gUfsgUfsuAfaCfscsA saAfL96 fsgAD-56741 A-115968.11 Gf(Geo)Uf(Teo)AfaC A-115861.11 uUfgaaGfuAfaAfuggU0.52 0.09 faCfCfAfuUf(Teo)Af fgUfuAfaCfcsasg (m5Ceo)Uf(Teo)Cf (Aeo)AfL96AD- A-113073.1 GfgUfuAfaCfaCfCfAf A-113074.1 uUfgAfaGfuAfaAfugg 0.930.10 0.63 0.10 54944/ uUfuAfcUfuCfaAfL96 UfgUfuAfaCfcsAfsg parentAD-56743 A-115965.5 Gf(Geo)UfuAfaCfaCf A-115861.11 uUfgaaGfuAfaAfuggU0.71 0.24 CfAfuUfuAfcUf(Teo) fgUfuAfaCfcsasg Cf(Aeo)AfL96 AD-56836.2A-115966.25 Gf(Geo)Uf(Teo)AfaC A-116284.3 uUfgAfaGf(Uhd)AfaA 1.01 0.110.73 0.11 faCfCfAfuUfuAfcUf fuggUfgUfuAfaCfcsa (Teo)Cf(Aeo)AfL96 sgAD-56797.2 A-115968.8 Gf(Geo)Uf(Teo)AfaC A-116250.6 uUfgAfaGfuAfaAfugg0.87 0.25 0.74 0.25 faCfCfAfuUf(Teo)Af Uf(Geo)UfuAfaCfcs(m5Ceo)Uf(Teo)Cf (Aeos)(Geo) (Aeo)AfL96 AD-56801.2 A-116277.6Gf(Geo)UfsuAfaCfsa A-116279.9 uUfsgAfaGfuAfaAfug 0.83 0.08 0.77 0.08CfsCfsAfuUfsuAfcUf gUfsgUfsuAfaCfscs suCfs(Aeo)AfL96 (Aeos)(Geo)AD-56831.2 A-116290.3 Gf(Geo)Uf(Teo)AfaC A-115962.21 uUfgAfaGfuAfaAfugg1.11 0.09 0.78 0.09 faCfCfAfuUf(Uhd)Af UfgUfuAfaCfcs cUf(Teo)Cf(Aeo)AfL(Aeos)(Geo) 96 AD-56830.2 A-116283.3 Gfs(Geo)UfsuAfaCfs A-116279.12uUfsgAfaGfuAfaAfug 1.01 0.13 0.79 0.13 aCfCfAfuUfsuAfcUfsgUfsgUfsuAfaCfscs uCfs(Aeo)AfL96 (Aeos)(Geo) AD-56761.2 A-116247.13gguuaacaCfCfAfuuua A-116244.6 uUfgaaGfuAfaAfuggU 0.82 0.26 0.83 0.26cu(Uhd)caaL96 fguuaaccsasg AD-56735.2 A-115966.29 Gf(Geo)Uf(Teo)AfaCA-115962.18 uUfgAfaGfuAfaAfugg 1.20 0.15 0.90 0.15 faCfCfAfuUfuAfcUfUfgUfuAfaCfcs (Teo)Cf(Aeo)AfL96 (Aeos)(Geo) AD-56872 A-115965.5Gf(Geo)UfuAfaCfaCf A-116392.1 uUfgaaGfuAfaAfuggU 0.93 0.24CfAfuUfuAfcUf(Teo) fguuAfaCfcs(Aeos) Cf(Aeo)AfL96 (Geo) AD-56820.2A-116317.4 gUf(Uhd)AfaCfaCfCf A-116297.10 uUfgAfaGfuAfaAfugg 1.05 0.100.94 0.10 AfuUfuAfcUfuCfaAfL UfgUfuAfaCfs 96 (m5Ceos)(Aeo) AD-56793.2A-116382.1 gdGuudAdAcadCdCauu A-116373.11 udTdGadAgdTadAdAug 0.98 0.080.99 0.08 dTdAcu(Uhd)dCadAL gdTdGuudAdAcdCsasg 96 PBS/ 1.00 0.04 1.000.04 Control AD-56873 A-115968.11 Gf(Geo)Uf(Teo)AfaC A-116392.1uUfgaaGfuAfaAfuggU 1.01 0.18 faCfCfAfuUf(Teo)Af fguuAfaCfcs(Aeos)(m5Ceo)Uf(Teo)Cf (Geo) (Aeo)AfL96 AD-54965 A-113033.1 AfaCfuGfcCfgAfCfUfA-113034.1 uUfuUfcGfaUfaGfagu 0.89 0.07 1.03 0.07 cUfaUfcGfaAfaAfL96CfgGfcAfgUfusCfsa AD-56787.2 A-116381.1 gguuaacaccdAudTudA A-116373.10udTdGadAgdTadAdAug 1.03 0.18 1.04 0.18 cdT(Uhd)dCasaL96gdTdGuudAdAcdCsasg AD-56840.2 A-116317.5 gUf(Uhd)AfaCfaCfCf A-116344.5uUfgAfaGfuAfaAfugg 1.38 0.14 1.05 0.14 AfuUfuAfcUfuCfaAfLUfgUfuAfaCfs(Chds) 96 (Aeo) AD-56834.2 A-116333.3 GbguuAfaCfaCfCfAfuA-116334.3 uUfgaaGfuAfaAfuggU 1.17 0.14 1.06 0.14 UfuAfcUfuCfaAfL96fguuAfaCfcsasGb AD-56841.2 A-115966.26 Gf(Geo)Uf(Teo)AfaC A-116285.3uUfgAfaGf(Uhd)AfaA 1.15 0.21 1.09 0.21 faCfCfAfuUfuAfcUffuggUfgUfuAfaCfcs (Teo)Cf(Aeo)AfL96 (Aeos)(Geo)

TABLE 15 AD-56813 optimized sequences and protein and activity levels.(The “Sense Sequence” column sequences are disclosed asSEQ ID NOS 941-959, respectively, in order of appearance,and the “Antisense Sequence” column sequences are disclosedas SEQ ID NOS 960-978, respectively, in order of appearance) ELISAActivity Duplex Sense Sense Antisense 10 mpk 3 mpk 10 mpk 3 mpk name ID(5′ to 3′) AS ID (5′ to 3′) Av SD Av SD Av SD Av SD AD- A- GfsgsUfuAf A-usUfsgAfaG 0.098 0.021 0.264 0.032 0.099 0.021 0.255 0.030 57213.1116858.1 aCfaCfCfAf 116861.1 fuAfaAfugg uUfuAfcUfu UfgUfuAfaC CfaAfL96fcsasg AD- A- GfsgsUfuAf A- usUfsgAfaG 0.140 0.027 0.200 0.132 0.1210.028 0.123 0.114 57214.1 116859.1 aCfaCfCfAf 116861.1 fuAfaAfugguUf(Uhd)Af UfgUfuAfaC cUfuCfaAfL fcsasg 96 AD- A- GfgUfuAfaC A-usUfsgAfaG 0.147 0.100 0.592 0.094 0.145 0.043 0.542 0.139 57205.1113073.1 faCfCfAfuU 116861.1 fuAfaAfugg fuAfcUfuCf UfgUfuAfaC aAfL96fcsasg AD- A- Gfs(Geo)Uf A- usUfsgAfaG 0.161 0.047 0.500 0.033 0.1790.055 0.583 0.065 56813.2 116280.6 suAfaCfsaC 116278.6 fuAfaAfuggfsCfsAfuUf UfsgUfsuAf suAfcUfsuC aCfscs fs(Aeo)AfL (Aeos) 96 (Geo) AD-A- GfgUfsuAfa A- usUfsgAfaG 0.164 0.026 0.355 0.007 0.198 0.011 0.3950.023 57212.1 116276.12 CfsaCfsCfs 116860.1 fuAfaAfugg AfuUfsuAfcUfsgUfsuAf UfsuCfsaAf aCfscsasg L96 AD- A- GfgUfuAfaC A- usUfsgAfaG0.170 0.127 0.659 0.004 0.121 0.094 0.608 0.060 57204.1 113073.1faCfCfAfuU 116860.1 fuAfaAfugg fuAfcUfuCf UfsgUfsuAf aAfL96 aCfscsasgAD- A- Gf(Geo)Uf A- uUfgaaGfuA 0.347 0.098 0.666 0.036 0.419 0.126 0.8800.070 56741.2 115968 (Teo)AfaCf 115861 faAfuggUfg aCfCfAfuUf UfuAfaCfcs(Teo)Af asg (m5Ceo)Uf (Teo)Cf (Aeo)AfL96 AD- A- GfgUfuAfaC A- usUfsgAfaG0.359 0.053 0.829 0.056 0.358 0.064 0.796 0.030 57203.1 113073.1faCfCfAfuU 116278.6 fuAfaAfugg fuAfcUfuCf UfsgUfsuAf aAfL96 aCfscs(Aeos) (Geo) AD- A- GfgUfuAfaC A- uUfsgAfaGf 0.366 0.053 0.668 0.0400.385 0.041 0.791 0.047 56765.2 113073.1 faCfCfAfuU 116275.7 uAfaAfuggUfuAfcUfuCf fsgUfsuAfa aAfL96 CfscsAfsg AD- A- GfgUfsuAfa A- uUfsgAfaGf0.401 0.057 0.700 0.059 0.406 0.078 0.785 0.059 56789.2 116276.12CfsaCfsCfs 116275.7 uAfaAfuggU AfuUfsuAfc fsgUfsuAfa UfsuCfsaAfCfscsAfsg L96 AD- A- GfsgsUfuAf A- uUfgAfaGfu 0.446 0.023 0.760 0.1420.536 0.027 0.831 0.117 57211.1 116858.1 aCfaCfCfAf 113074.1 AfaAfuggUfuUfuAfcUfu gUfuAfaCfc CfaAfL96 sAfsg AD- A- GfsgsUfuAf A- uUfgAfaGfu0.476 0.028 0.734 0.053 0.547 0.041 0.826 0.065 57210.1 116857.1aCfaCfsCfs 113074.1 AfaAfuggUf AfuUfuAfcU gUfuAfaCfc fuCfaAfL96 sAfsgAD- A- GfgUfsuAfa A- uUfgAfaGfu 0.542 0.016 0.915 0.085 0.486 0.0060.831 0.034 57208.1 116276.12 CfsaCfsCfs 113074.1 AfaAfuggUf AfuUfsuAfcgUfuAfaCfc UfsuCfsaAf sAfsg L96 AD- A- Gf(Geo)Uf A- uUfgAfaGfu 0.5740.058 0.932 0.127 0.415 0.019 0.735 0.123 56475.3 115968 (Teo)AfaCf113074.1 AfaAfuggUf aCfCfAfuUf gUfuAfaCfc (Teo)Af sAfsg (m5Ceo)Uf(Teo)Cf (Aeo)AfL96 AD- A- Gfs(Geo)Uf A- uUfgAfaGfu 0.588 0.135 0.9660.100 0.460 0.136 0.823 0.026 57209.1 116280.6 suAfaCfsaC 113074.1AfaAfuggUf fsCfsAfuUf gUfuAfaCfc suAfcUfsuC sAfsg fs(Aeo)AfL 96 AD- A-GfgUfuAfaC A- uUfgAfaGfu 0.617 0.025 0.715 0.071 0.667 0.016 0.835 0.08354944.13 113073.1 faCfCfAfuU 113074.1 AfaAfuggUf fuAfcUfuCf gUfuAfaCfcaAfL96 sAfsg AD- A- GfgUfuAfaC A- uUfgAfaGfu 0.681 0.097 0.811 0.1100.707 0.068 0.940 0.149 57206.1 113073.1 faCfCfAfuU 116870.1 AfaAfuggUffuAfcUfuCf gUfuAfaCfc aAfL96 Afsg AD- A- GfgUfsuAfa A- uUfgAfaGfu 0.7360.109 0.714 0.101 0.729 0.122 0.817 0.090 57208.2 116276.12 CfsaCfsCfs113074.1 AfaAfuggUf AfuUfsuAfc gUfuAfaCfc UfsuCfsaAf sAfsg L96 AD- A-GfgUfuAfaC A- uUfgAfaGfu 0.934 0.043 0.975 0.035 0.984 0.104 1.009 0.06657207.1 113073.1 faCfCfAfuU 116871.1 AfaAfuggUf fuAfcUfuCf gUfuAfaCfc1.000 0.029 1.000 1.000 0.077 1.000 aAfL96 Afg

Example 8 Serpinc1 Knock-Down in Hemophilic Mice

Male and female mice having a targeted deletion of Factor VIII(C57BL/6/129 hybrids) and recapitulating the hemophilia A phenotype andcontrol or wild-type (C57BL/6 female) mice were subcutaneously injectedwith a single dose of compound AD-57213 conjugated to GalNAc at 30mg/kg, 10 mg/kg, 3 mg/kg, or 1 mg/kg at Day 0, animals were sacrificedat Day 3 and Serpinc1 activity was determined as described above. FIG.13 shows that, not only does a single dose of AD-57213 effectivelyknock-down Serpinc1 activity, but there is also a dose response toAD-57213.

To investigate the impact of antithrombin reduction on thrombingeneration in a hemophilia setting, thrombin generation studies wereperformed on Factor IX (FIX) and Anthithrombin- (AT-) depleted humanplasma. FIX depletion recapitulates the hemophilia B phenotype AT wassubsequently added back to the plasma samples at various levels (1IU/ml=100%) to generate FIX-depleted plasma samples with differentlevels of antithrombin (0-100%). Control plasma was generated by addingback 1 IU/ml antithrombin and 5 μg/ml FIX (100%) to the double-depletedplasma. FIG. 31A depicts thrombin generation in FIX- and AT-depletedhuman plasma (tissue factor=5 pM). FIG. 31B depicts the peak thrombin inFIX- and AT-depleted human plasma (tissue factor=5 pM). Therefore,antithrombin reduction increases thrombin generation in FactorIX-depleted human plasma in vitro.

Example 9 Dose Duration of AD-57213

In order to evaluate the duration of anti-thrombin silencing inHemophilia A mice (B6;129S4-F8^(tm1/Kaz/J); Jackson Labs) following asingle dose of AD-57213 conjugated to GalNAc, mice were subcutaneouslyinjected with compounds AD-57214, AD-57205, or AD-57213 or PBS. Wholeblood was collected retroorbitally and assayed for Serpinc1 mRNA levelsand Serpinc1 activity. The results of the single dose screen forcompound AD-57213 administered at 10 mg/kg, 3 mg/kg, or 1 mg/kg as apercent knock-down of Serpinc1 activity from PBS at Days 3, 7, 10, 14,17, 21, 28, and 36 are depicted in FIG. 14. FIG. 16 shows the results ofthe single dose screen for compounds AD-57213, AD-57205, and AD-57214administered at the doses indicated in the Figures as a percentknock-down of Serpinc1 activity from PBS at Days 3, 7, 10, 14, 17, 21,and 25.

Liver mRNA, AT antigen in serum and AT activity were measured inhemophilia A mice (B6;129S4-F8^(tm1/Kaz/J); Jackson labs) injectedsubcutaneously with AD-57213 at a dose of 30 mg/kg, 10 mg/kg, 3 mg/kg, 1mg/kg, or PBS at Day 0. Animals were sacrificed on Day 3 post-injectionas described above. FIG. 15 shows the results of the single dose screenas a percent knock-down of Serpinc1 mRNA levels from PBS, as a percentknock-down of Serpinc1 antigen levels from PBS, and as a percentknock-down of Serpinc1 activity from PBS at Day 3 for AD-57213.

As evidenced by FIGS. 14-16, administration of compound AD-57213 leadsto potent, dose-dependent suppression of Serpinc1 in HA mice with asingle dose ED₅₀ of less than 1 mg/kg on Day 7. Serpinc1 suppression wasdurable and correlated with the maximal level of antithrombinsuppression achieved. A single dose of 1 mg/kg led to the maintenance of50% suppression for about 15 days, while a dose of 10 mg/kg led togreater than 80% suppression maintained for 28 days.

Example 10 Dose Duration of a Split-Dose of AD-57213

In order to further evaluate compound AD-57213 knock-down of Serpinc1expression and activity, a split-dosing experiment was performed.C57BL/6 mice were subcutaneously administered GalNAc-conjugated AD-57213and the effect of a 3 times per week, 1/3 dose of AD-57213 was comparedto the effect of a 1 time per week fully concentrated dose of AD-57213.A summary of the study design is presented in Table 16. Serum Serpinc1protein levels were determined.

TABLE 16 Study Design of Split-Dosing Experiment Group Test compoundDose (mg/kg) Frequency 1 AD-57213 3 q1w 2 1.5 (Monday) 3 0.75 4 1 t.i.w.5 0.5 (M, W, F) 6 0.25 7 PBS — q1w (q1w: Once a week)

The results of the once a week (q1w) dosing as a percent knock-down ofSerpinc1 protein levels from PBS are shown in FIG. 17 and the results ofthe three-time per week (t.i.w.) dosing as a percent knock-down ofSerpinc1 protein levels from PBS are shown in FIG. 18.

As shown in FIGS. 17 and 18, repeat dosing of compound AD-57213 led to adose-dependent, durable response, with some additive effect. Animalsdosed with 3 mg/kg reached the nadir levels of >95% knock-down after 2weekly doses whereas the lower two dose groups attained nadir after 3weekly doses (˜90% knock-down for 1.5 mg/kg and ˜80% for 0.75 mg/kg).

To further study the different dosing regimens, in different groups, thesame weekly dose was split and dosed three times a week, e.g., 1.5 mg/kgq1w was compared with 0.5 mg/kg tiw. As shown in FIG. 19, the cumulativeweekly dose gave the same level of knock-down. For example, Serpinc1levels achieved with 1.5 mg/kg (q1w) were equivalent to Serpinc1 levelsachieved with 0.5 mg/kg administered three times a week.

Example 11 Non-Human Primate Dosing of Serpinc1 siRNAs

Compound AD-57213 was tested for efficacy in non-human primates asoutlined in Table 17. Serum Serpinc1 protein levels were determined atDays −14, −8, −4, Day 1 at 4 hours post-dosing, Days 2, 4, 8, 11, 15,22, 29, 37, 44, 51, and 58.

TABLE 17 Non-Human Primate Dosing Experiment. Test Group Dose levelRoute of Article Number n (mg/kg) administration Rationale AD54944 1 310 SC Parent compound AD-57213  2* 3 30 SC Dose response Similar to 3 ×for the lead 10 mpk 3 3 10 SC Compare compounds at 10 mpk 4 3 3 SC Dosecurve 5 3 1 SC AD-57205 5 3 10 SC Same potency Has less PS as 57213 at10 mpk in mice LNP- 7 3 0.3 IV Test target Positive control 55029 fortarget knock-down and assays

FIG. 20 shows the results of the single dose screen for all compoundstested as a group average of the relative serum Serpinc1 levels comparedto pre-dosing Serpinc1 levels and demonstrates that all of the siRNAstested effectively knock-down Serpinc1 protein levels.

FIG. 21 shows the results of the single dose screen for compoundAD-57213 as a group average of the relative serum Serpinc1 levelscompared to pre-dosing Serpinc1 levels.

FIG. 22 shows the results of the single dose screen for all compoundstested as a group average of the relative serum Serpinc1 levels comparedto pre-dosing Serpinc1 levels on Day 8.

Overall, the results demonstrate that there is dose-dependent knock-downof Serpinc1 protein levels with AD-57213 in non-human primates and thatboth AD-57213 and AD-57205 show improved potency over the parentcompound AD-52444.

Example 12 Non-Human Primate Dosing of a Therapeutic Serpinc1 siRNA

Compound AD-57213 was tested for efficacy in non-human primates.Cynomolgus monkeys were administered compound AD-57213 as outlined inTable 18 below. Plasma was collected at various time points afteradministration of AD-57213 and analyzed for antithrombin protein(Serpinc1) levels by ELISA.

TABLE 18 Study Design: AD-57213 pharmacology in nonhuman primates Routeof Group Dose Level Adminis- Sample Number Test Article N (mg/kg)tration Collection 1 AD-57213 3 1 SC Plasma for AT 2 3 3 SC protein and3 3 10 SC thrombin 4 3 30 SC generation

FIG. 23 shows the results of the single dose screen for compoundAD-57213 as a group average of the relative serum Serpinc1 levelscompared to the average of three pre-dose measurements. The resultsdemonstrate dose dependent Serpinc1 silencing with approximately 50, 70,80 and >90% silencing at 1, 3, 10 and 30 mg/kg, respectively. Datapoints represent group mean and error bars represent standard deviation.

FIGS. 24A-D show the relationship between relative serum Serpinc1 leveland fold change in peak plasma thrombin level at a single A) 1 mg/kg, B)3 mg/kg, C) 10 mg/kg, and D) 30 mg/kg dose of compound AD57213. Serpinc1levels are represented relative to the average of three pre-dosemeasurements. Thrombin generation curves were generated from plasmasamples collected at various time points using a Calibrated AutomatedThrombinoscope (tissue factor=1 pM). Fold change in peak thrombin wascalculated relative to the average peak thrombin value for two pre-dosevalues for each animal. Data points represent group mean and error barsrepresent standard deviation. FIG. 25 shows a consolidated scatterplotof fold change increase in peak thrombin as a function of relativeSerpinc1 silencing.

Animals were also administered three weekly AD-57213 doses of 30 mg/kgand the Serpinc1 protein and mRNA levels were determined in bloodsamples collected from the animals. The results of these studies arepresented in FIGS. 26A (Serpinc1 protein levels relative to prebleedlevels) and 26B (Serpinc1 mRNA levels relative to GAPDH).

Overall, the results demonstrate that there is a durable, dose-dependentinhibition of Serpinc1 protein levels with compound AD-57213 innon-human primates that results in up to a 4-fold increase in thrombingeneration.

Example 13 Repeat Administration of a Serpinc1 SiRNA in Non-HumanPrimate Dosing

Compound AD-57213 was tested for efficacy and to evaluate the cumulativeeffect of the compound in non-human primates with a repeatadministration protocol. Cynomolgus monkeys were administered compoundAD-57213 at 0.5 mg/kg q1w; 1 mg/kg q2w (every other week), for 2 monthsand 1.5 mg/kg q1w; 3 mg/kg q2w for 6 weeks. Serum was collected atvarious time points as illustrated in FIG. 27 and analyzed forantithrombin protein level (SerpinC1) by ELISA. Antithrombin levels wererepresented relative to the average of three pre-dose measurements.

The first two dose groups with 0.5 mg/kg weekly cumulative dose led to80% decrease in AT levels after 5 weeks. The latter two groups with 1.5mg/kg the cumulative weekly dose led to >95% maximum knockdown. FIG. 27Ashows the data from latter two groups. Animals receiving the 3 mg/kgdose were euthanized on Day 54 and animals receiving the 1.5 mg/kg q1wdose were administered an additional 6th weekly dose on day 36 and arebeing monitored for recovery to base line levels. FIG. 27B shows ATlevels after 0.5 mg/kg cumulative weekly dose.

The data demonstrate that dose dependent antithrombin silencing wasobserved with all dosing regimens and achieved a steady-state level ofsuppression by day 25.

Example 14 Correction in Hemostasis Following Administration of CompoundAD-57213 in Hemophilic Mice

Hemophilic animals have less thrombin generation potential and cannotform stable clots. Reduction of antithrombin protein in these animalsshould help rebalance the hemostasis, increase the endogenous thrombingenerating potential, and enable clot formation. This hypothesis wastested in hemophilia A and hemophilia B mice in the microvessel laserinjury model accompanied with intravital imaging. Mice were injectedwith compound AD-57213 and injury was induced 10 days post-treatment.Accumulation of platelets and fibrin at the site of injury werevisualized, recorded and quantified. FIG. 28A shows the median values ofplatelet accumulation over time after laser surgery and FIG. 28B showsthe median fibrin values from all inflicted injuries over time afterlaser surgery. As demonstrated by FIGS. 28A and 28B, compound AD-57213injected at 1 mg/kg or 30 mg/kg led to platelet and fibrin depositionleading to clot formation in 100% of the injuries. Table 19 summarizesthe results from two separate experiments with HA and HB animals.

TABLE 19 Animals Injuries Stable Percent AT Group (N) (N) Thrombus (N)mRNA in liver WT 2 10 10 100% HA + PBS 2 13 0 100% HA + 1 mg/kg 4 20 20 50% ALN-AT3 HA + 30 mg/kg 4 20 20  5% ALN-AT3 HB + PBS 2 6 0 100% HB +30 mg/kg 2 6 6  5% ALNAT3

Example 15 In Vivo Efficacy of AD-57213 LNP Formulation

Compounds AD-55029 (unconjugated) and AD-57213 (conjugated to GalNAc)were formulated in a lipid nucleic acid particle (AF-11) and wild-typeanimals were administered doses of 0.03 mg/kg, 0.1 mg/kg and 0.3 mg/kgof these LNP formulated compounds. Luciferase (AF11-1955) was used ascontrol.

Both compounds led to >95% knock-down at 0.3 mg/kg but the levels weremaintained by AF11-57213 for 15 days versus 8 days by AF11-55029 (seeFIG. 29). A similar difference in duration of action between the twocompounds was observed at the lower doses.

Example 16 Design, Synthesis, and In Vitro Screening of AdditionalsiRNAs

siRNA Design

SiRNA duplexes, 19 nucleotides long for both the sense and antisensestrand, were designed using the human SERPINC1 mRNA sequence set forthin GenBank Accession No.

NM_(—)000488.3. One thousand five hundred and eighty-one duplexes wereinitially identified that did not contain repeats longer than 7nucleotides, spanning the entire 1599 nucleotide transcript. All 1581duplexes were then scored for predicted efficacy according to a linearmodel that evaluates the nucleotide pair at each duplex position, andthe dose and cell line to be used for screening. The duplexes were alsomatched against all transcripts in the human RefSeq collection using acustom brute force algorithm, and scored for lowest numbers ofmismatches (per strand) to transcripts other than SERPINC1. Duplexes tobe synthesized and screened were then selected from the 1581, accordingto the following scheme: Beginning at the 5′ end of the transcript, aduplex was selected within a “window” of every 10±2 nucleotides that

1) had the highest predicted efficacy,

2) had at least one mismatch in both strands to all transcripts otherthan SERPINC1,

3) had not already been synthesized and screened as part of other duplexsets.

If no duplex could be identified within a given window that satisfiedall criteria, that window was skipped. One hundred and sixty-fourduplexes were identified that satisfied these criteria.

A detailed list of Sepinc1 sense and antisense strand sequences is shownin Tables 20 and 21.

TABLE 20 AT3 (SERPINC1) unmodified sequences (The “Sense Sequence”column sequences are disclosed as SEQ ID NOS 979-1141,respectively, in order of appearance, and the “Antisense Sequence”column sequences are disclosed asSEQ ID NOS 1142-1304, respectively, in order of appearance) Sense AntisDuplex Oligo Oligo Position in Name Name Sense Sequence NameAntisense Sequence NM_000488.3 AD-59267.1 A-120250.1 GGAGAAGAAGGCAACUGAGA-120251.1 CUCAGUUGCCUUCUUCUCC 293-311 AD-59268.1 A-120266.1UGACCAAGCUGGGUGCCUG A-120267.1 CAGGCACCCAGCUUGGUCA 481-499 AD-59269.1A-120282.1 GGUUAACACCAUUUACUUC A-120283.1 GAAGUAAAUGGUGUUAACC 860-878AD-59270.1 A-120298.1 GCUGGUUAACACCAUUUAC A-120299.1 GUAAAUGGUGUUAACCAGC857-875 AD-59271.1 A-120314.1 UAAUGACACCCUCCAGCAA A-120315.1UUGCUGGAGGGUGUCAUUA 500-518 AD-59272.1 A-120330.1 CGUGUUCAGCAUCUAUGAUA-120331.1 AUCAUAGAUGCUGAACACG 952-970 AD-59273.1 A-120252.1UUGAGGACGGCUUCAGUUU A-120253.1 AAACUGAAGCCGUCCUCAA 1189-1207 AD-59274.1A-120268.1 CGGCGUGUCUGGGAACUGU A-120269.1 ACAGUUCCCAGACACGCCG 351-369AD-59275.1 A-120284.1 UUAACACCAUUUACUUCAA A-120285.1 UUGAAGUAAAUGGUGUUAA862-880 AD-59276.1 A-120300.1 CCCUGAAAAGUCCAAACUC A-120301.1GAGUUUGGACUUUUCAGGG 1250-1268 AD-59277.1 A-120316.1 CGAGAUGACCUCUAUGUCUA-120317.1 AGACAUAGAGGUCAUCUCG 1290-1308 AD-59278.1 A-120332.1UCUACAAGGCUGAUGGAGA A-120333.1 UCUCCAUCAGCCUUGUAGA 931-949 AD-59279.1A-120254.1 AGCUCACUGUUCUGGUGCU A-120255.1 AGCACCAGAACAGUGAGCU 841-859AD-59280.1 A-120270.1 AGGAGCAGCUGCAAGACAU A-120271.1 AUGUCUUGCAGCUGCUCCU1210-1228 AD-59281.1 A-120286.1 GCCACCAACCGGCGUGUCU A-120287.1AGACACGCCGGUUGGUGGC 342-360 AD-59282.1 A-120302.1 CAGAACAGAAGAUCCCGGAA-120303.1 UCCGGGAUCUUCUGUUCUG 322-340 AD-59283.1 A-120318.1CCUUGUCGAUCUGUUCAGC A-120319.1 GCUGAACAGAUCGACAAGG 1232-1250 AD-59284.1A-120334.1 AGGCAAGUUCCGUUAUCGG A-120335.1 CCGAUAACGGAACUUGCCU 980-998AD-59285.1 A-120256.1 UUUUGUCCUUGCUGCUCAU A-120257.1 AUGAGCAGCAAGGACAAAA172-190 AD-59286.1 A-120272.1 AGACCUACCAGGACAUCAG A-120273.1CUGAUGUCCUGGUAGGUCU 682-700 AD-59287.1 A-120288.1 AACUGAACUGCCGACUCUAA-120289.1 UAGAGUCGGCAGUUCAGUU 589-607 AD-59288.1 A-120304.1CAUUUACUUCAAGGGCCUG A-120305.1 CAGGCCCUUGAAGUAAAUG 869-887 AD-59289.1A-120320.1 CCCUGGACUUCAAGGAAAA A-120321.1 UUUUCCUUGAAGUCCAGGG 730-748AD-59290.1 A-120336.1 AGCUGCAAGUACCGCUGUU A-120337.1 AACAGCGGUACUUGCAGCU1361-1379 AD-59291.1 A-120258.1 ACACAAGGAAGGAACUGUU A-120259.1AACAGUUCCUUCCUUGUGU 913-931 AD-59292.1 A-120274.1 GCAACUGAGGAUGAGGGCUA-120275.1 AGCCCUCAUCCUCAGUUGC 303-321 AD-59293.1 A-120290.1GUAGCCAACCCUUGUGUUA A-120291.1 UAACACAAGGGUUGGCUAC 1491-1509 AD-59294.1A-120306.1 GUUUGUGAACAGAAGUAAA A-120307.1 UUUACUUCUGUUCACAAAC 1550-1568AD-59295.1 A-120322.1 GGGUGACUUUCAAGGCCAA A-120323.1 UUGGCCUUGAAAGUCACCC1411-1429 AD-59296.1 A-120338.1 UUAUCGGCGCGUGGCUGAA A-120339.1UUCAGCCACGCGCCGAUAA  992-1010 AD-59297.1 A-120260.1 CCACUUCUUCUUUGCCAAAA-120261.1 UUUGGCAAAGAAGAAGUGG 572-590 AD-59298.1 A-120276.1AACACCAUUUACUUCAAGG A-120277.1 CCUUGAAGUAAAUGGUGUU 864-882 AD-59299.1A-120292.1 GAUGGAGAGUCGUGUUCAG A-120293.1 CUGAACACGACUCUCCAUC 942-960AD-59300.1 A-120308.1 CACCAUUUACUUCAAGGGC A-120309.1 GCCCUUGAAGUAAAUGGUG866-884 AD-59301.1 A-120324.1 UUUACUUCAAGGGCCUGUG A-120325.1CACAGGCCCUUGAAGUAAA 871-889 AD-59302.1 A-120340.1 UAAGAGAAGUUCCUCUGAAA-120341.1 UUCAGAGGAACUUCUCUUA 1450-1468 AD-59303.1 A-120262.1GCGGGACAUUCCCAUGAAU A-120263.1 AUUCAUGGGAAUGUCCCGC 251-269 AD-59304.1A-120278.1 UGCCCCACCCUGUCCUCUG A-120279.1 CAGAGGACAGGGUGGGGCA  21-MarAD-59305.1 A-120294.1 UGGUUAACACCAUUUACUU A-120295.1 AAGUAAAUGGUGUUAACCA859-877 AD-59306.1 A-120310.1 CGGAUUGCCUCAGAUCACA A-120311.1UGUGAUCUGAGGCAAUCCG 62-80 AD-59307.1 A-120326.1 CCAGGACAUCAGUGAGUUGA-120327.1 CAACUCACUGAUGUCCUGG 689-707 AD-59308.1 A-120342.1CCAGUUUUCAGGCGGAUUG A-120343.1 CAAUCCGCCUGAAAACUGG 50-68 AD-59309.1A-120264.1 CACCAUAUCUGAGAAAACA A-120265.1 UGUUUUCUCAGAUAUGGUG 542-560AD-59310.1 A-120280.1 AAGUAAAAAUAAAUACAAA A-120281.1 UUUGUAUUUAUUUUUACUU1562-1580 AD-59311.1 A-120296.1 GCACCCAGGUGCUUGAGUU A-120297.1AACUCAAGCACCUGGGUGC 1012-1030 AD-59312.1 A-120312.1 UAACACCAUUUACUUCAAGA-120313.1 CUUGAAGUAAAUGGUGUUA 863-881 AD-59313.1 A-120328.1CCUUCAAAGGUGAUGACAU A-120329.1 AUGUCAUCACCUUUGAAGG 1033-1051 AD-59314.1A-120344.1 CAAGGCCAAUUCCCGCUUU A-120345.1 AAAGCGGGAAUUGGCCUUG 371-389AD-59315.1 A-120360.1 UCAGUUUGAAGGAGCAGCU A-120361.1 AGCUGCUCCUUCAAACUGA1201-1219 AD-59316.1 A-120376.1 GGGACUGCGUGACCUGUCA A-120377.1UGACAGGUCACGCAGUCCC 199-217 AD-59317.1 A-120392.1 UCAGCCAAUCGCCUUUUUGA-120393.1 CAAAAAGGCGAUUGGCUGA 639-657 AD-59318.1 A-120408.1CCUCGGAAGCCAUCAAUGA A-120409.1 UCAUUGAUGGCUUCCGAGG 823-841 AD-59319.1A-120424.1 GACAAUGAUAACAUUUUCC A-120425.1 GGAAAAUGUUAUCAUUGUC 429-447AD-59320.1 A-120346.1 CUUAUUCUUUGCACCUCUU A-120347.1 AAGAGGUGCAAAGAAUAAG1521-1539 AD-59321.1 A-120362.1 AUUGCUGGCCGUUCGCUAA A-120363.1UUAGCGAACGGCCAGCAAU 1383-1401 AD-59322.1 A-120378.1 AAAUGAAGAAGGCAGUGAAA-120379.1 UUCACUGCCUUCUUCAUUU 1340-1358 AD-59323.1 A-120394.1UGAGUUGGUAUAUGGAGCC A-120395.1 GGCUCCAUAUACCAACUCA 701-719 AD-59324.1A-120410.1 UCCAGCAACUGAUGGAGGU A-120411.1 ACCUCCAUCAGUUGCUGGA 511-529AD-59325.1 A-120426.1 AACUGUAACCUCUGGAAAA A-120427.1 UUUUCCAGAGGUUACAGUU140-158 AD-59326.1 A-120348.1 UCAACAAAUGGGUGUCCAA A-120349.1UUGGACACCCAUUUGUUGA 772-790 AD-59327.1 A-120364.1 GAUUAGCGGCCAUGUAUUCA-120365.1 GAAUACAUGGCCGCUAAUC 109-127 AD-59328.1 A-120380.1GUGCUUGAGUUGCCCUUCA A-120381.1 UGAAGGGCAACUCAAGCAC 1020-1038 AD-59329.1A-120396.1 UGCAGAAGGCCGAGAUGAC A-120397.1 GUCAUCUCGGCCUUCUGCA 1280-1298AD-59330.1 A-120412.1 CUAUUUUUGGUUUGUGAAC A-120413.1 GUUCACAAACCAAAAAUAG1541-1559 AD-59331.1 A-120428.1 CAGUGAAGCAGCUGCAAGU A-120429.1ACUUGCAGCUGCUUCACUG 1352-1370 AD-59332.1 A-120350.1 GUGUCCAAUAAGACCGAAGA-120351.1 CUUCGGUCUUAUUGGACAC 783-801 AD-59333.1 A-120366.1AUGAAUUGGAGGAGAUGAU A-120367.1 AUCAUCUCCUCCAAUUCAU 1141-1159 AD-59334.1A-120382.1 AUCUAUGAUGUACCAGGAA A-120383.1 UUCCUGGUACAUCAUAGAU 962-980AD-59335.1 A-120398.1 CCAUAAGGCAUUUCUUGAG A-120399.1 CUCAAGAAAUGCCUUAUGG1319-1337 AD-59336.1 A-120414.1 AUGCAUUCCAUAAGGCAUU A-120415.1AAUGCCUUAUGGAAUGCAU 1312-1330 AD-59337.1 A-120430.1 UGGUGCUGGUUAACACCAUA-120431.1 AUGGUGUUAACCAGCACCA 853-871 AD-59338.1 A-120352.1AUCUGUUCAGCCCUGAAAA A-120353.1 UUUUCAGGGCUGAACAGAU 1240-1258 AD-59339.1A-120368.1 AGAUGAUGCUGGUGGUCCA A-120369.1 UGGACCACCAGCAUCAUCU 1153-1171AD-59340.1 A-120384.1 AUAUGGAGCCAAGCUCCAG A-120385.1 CUGGAGCUUGGCUCCAUAU710-728 AD-59341.1 A-120400.1 CCGAAUCACCGAUGUCAUU A-120401.1AAUGACAUCGGUGAUUCGG 803-821 AD-59342.1 A-120416.1 GCAGAGCAAUCCAGAGCGGA-120417.1 CCGCUCUGGAUUGCUCUGC 750-768 AD-59343.1 A-120432.1ACACCAUUUACUUCAAGGG A-120433.1 CCCUUGAAGUAAAUGGUGU 865-883 AD-59344.1A-120354.1 GUACCAGGAAGGCAAGUUC A-120355.1 GAACUUGCCUUCCUGGUAC 971-989AD-59345.1 A-120370.1 UGCCCAAGCCUGAGAAGAG A-120371.1 CUCUUCUCAGGCUUGGGCA1069-1087 AD-59346.1 A-120386.1 UUCUUUGCCAAACUGAACU A-120387.1AGUUCAGUUUGGCAAAGAA 579-597 AD-59347.1 A-120402.1 UGGCCAAGGUAGAGAAGGAA-120403.1 UCCUUCUCUACCUUGGCCA 1090-1108 AD-59348.1 A-120418.1UGCUGCAAGAGUGGCUGGA A-120419.1 UCCAGCCACUCUUGCAGCA 1123-1141 AD-59349.1A-120434.1 CCAUGUGCAUUUACCGCUC A-120435.1 GAGCGGUAAAUGCACAUGG 271-289AD-59350.1 A-120356.1 AGACAUGGGCCUUGUCGAU A-120357.1 AUCGACAAGGCCCAUGUCU1223-1241 AD-59351.1 A-120372.1 UUUUGGAGACAAAUCCCUU A-120373.1AAGGGAUUUGUCUCCAAAA 653-671 AD-59352.1 A-120388.1 GGAUGAGGGCUCAGAACAGA-120389.1 CUGUUCUGAGCCCUCAUCC 311-329 AD-59353.1 A-120404.1CGGCUUUUGCUAUGACCAA A-120405.1 UUGGUCAUAGCAAAAGCCG 469-487 AD-59354.1A-120420.1 AGCUCCAGCCCCUGGACUU A-120421.1 AAGUCCAGGGGCUGGAGCU 721-739AD-59355.1 A-120436.1 CUGAUCAGAUCCACUUCUU A-120437.1 AAGAAGUGGAUCUGAUCAG562-580 AD-59356.1 A-120358.1 UGCUGGUGGUCCACAUGCC A-120359.1GGCAUGUGGACCACCAGCA 1159-1177 AD-59357.1 A-120374.1 GCGAGAUUUAGAGGAAAGAA-120375.1 UCUUUCCUCUAAAUCUCGC 30-48 AD-59358.1 A-120390.1CUGCUCAUUGGCUUCUGGG A-120391.1 CCCAGAAGCCAAUGAGCAG 183-201 AD-59359.1A-120406.1 CCUUCAAUGAGACCUACCA A-120407.1 UGGUAGGUCUCAUUGAAGG 673-691AD-59360.1 A-120422.1 ACCAUUUACUUCAAGGGCC A-120423.1 GGCCCUUGAAGUAAAUGGU867-885 AD-59587.1 A-120438.1 GCACCUCUUCCUAUUUUUG A-120439.1CAAAAAUAGGAAGAGGUGC 1531-1549 AD-59588.1 A-120454.1 GUGGCUGAAGGCACCCAGGA-120455.1 CCUGGGUGCCUUCAGCCAC 1002-1020 AD-59589.1 A-120470.1UCCGCAUUGAGGACGGCUU A-120471.1 AAGCCGUCCUCAAUGCGGA 1183-1201 AD-59590.1A-120486.1 UGGACAUCUGCACAGCCAA A-120487.1 UUGGCUGUGCAGAUGUCCA 229-247AD-59591.1 A-120502.1 GUCCAAACUCCCAGGUAUU A-120503.1 AAUACCUGGGAGUUUGGAC1259-1277 AD-59592.1 A-120518.1 AGAAGGAACUCACCCCAGA A-120519.1UCUGGGGUGAGUUCCUUCU 1102-1120 AD-59593.1 A-120440.1 UCUUGAGGUAAAUGAAGAAA-120441.1 UUCUUCAUUUACCUCAAGA 1331-1349 AD-59594.1 A-120456.1AGCCCUGUGGACAUCUGCA A-120457.1 UGCAGAUGUCCACAGGGCU 222-240 AD-59595.1A-120472.1 CAGAGCGGCCAUCAACAAA A-120473.1 UUUGUUGAUGGCCGCUCUG 761-779AD-59596.1 A-120488.1 AUUUAAGUUUGACACCAUA A-120489.1 UAUGGUGUCAAACUUAAAU530-548 AD-59597.1 A-120504.1 UGAGAAGAGCCUGGCCAAG A-120505.1CUUGGCCAGGCUCUUCUCA 1079-1097 AD-59598.1 A-120520.1 UCACCAUGGUCCUCAUCUUA-120521.1 AAGAUGAGGACCAUGGUGA 1051-1069 AD-59599.1 A-120442.1GGAAGGAACUGUUCUACAA A-120443.1 UUGUAGAACAGUUCCUUCC 919-937 AD-59600.1A-120458.1 CUGGUUUUUAUAAGAGAAG A-120459.1 CUUCUCUUAUAAAAACCAG 1440-1458AD-59601.1 A-120474.1 CUGGGUGCCUGUAAUGACA A-120475.1 UGUCAUUACAGGCACCCAG489-507 AD-59602.1 A-120490.1 GUACCGCUGUUGUGAUUGC A-120491.1GCAAUCACAACAGCGGUAC 1369-1387 AD-59603.1 A-120506.1 UCUAUCAGCACCUGGCAGAA-120507.1 UCUGCCAGGUGCUGAUAGA 400-418 AD-59604.1 A-120522.1CUGGCAGAUUCCAAGAAUG A-120523.1 CAUUCUUGGAAUCUGCCAG 411-429 AD-59605.1A-120444.1 CGAUGUCAUUCCCUCGGAA A-120445.1 UUCCGAGGGAAUGACAUCG 812-830AD-59606.1 A-120460.1 GCUUCUGGGACUGCGUGAC A-120461.1 GUCACGCAGUCCCAGAAGC193-211 AD-59607.1 A-120476.1 CCUGUCACGGGAGCCCUGU A-120477.1ACAGGGCUCCCGUGACAGG 211-229 AD-59608.1 A-120492.1 AUUUACUUCAAGGGCCUGUA-120493.1 ACAGGCCCUUGAAGUAAAU 870-888 AD-59609.1 A-120508.1UGCUACCACUUUCUAUCAG A-120509.1 CUGAUAGAAAGUGGUAGCA 389-407 AD-59610.1A-120524.1 GGAACUGUCCAAGGCCAAU A-120525.1 AUUGGCCUUGGACAGUUCC 362-380AD-59611.1 A-120446.1 ACAAAUCCUCCAAGUUAGU A-120447.1 ACUAACUUGGAGGAUUUGU619-637 AD-59612.1 A-120462.1 AUUUACCGCUCCCCGGAGA A-120463.1UCUCCGGGGAGCGGUAAAU 279-297 AD-59613.1 A-120478.1 ACCCCUGAGUAUCUCCACGA-120479.1 CGUGGAGAUACUCAGGGGU 452-470 AD-59614.1 A-120494.1CACUAUCUCCACUUGCCCA A-120495.1 UGGGCAAGUGGAGAUAGUG 79-97 AD-59615.1A-120510.1 AAAUACAAACUACUUCCAU A-120511.1 AUGGAAGUAGUUUGUAUUU 1572-1590AD-59616.1 A-120526.1 CUGGUUAACACCAUUUACU A-120527.1 AGUAAAUGGUGUUAACCAG858-876 AD-59617.1 A-120448.1 UCAUCUUGCCCAAGCCUGA A-120449.1UCAGGCUUGGGCAAGAUGA 1063-1081 AD-59618.1 A-120464.1 CCUCAGAUCACACUAUCUCA-120465.1 GAGAUAGUGUGAUCUGAGG 69-87 AD-59619.1 A-120480.1CUGUCCUCUGGAACCUCUG A-120481.1 CAGAGGUUCCAGAGGACAG  30-Dec AD-59620.1A-120496.1 CCCUGUGGAAGAUUAGCGG A-120497.1 CCGCUAAUCUUCCACAGGG  99-117AD-59621.1 A-120512.1 AUCUCCACGGCUUUUGCUA A-120513.1 UAGCAAAAGCCGUGGAGAU462-480 AD-59622.1 A-120528.1 GUGGCUGGAUGAAUUGGAG A-120529.1CUCCAAUUCAUCCAGCCAC 1133-1151 AD-59623.1 A-120450.1 CAAGUUAGUAUCAGCCAAUA-120451.1 AUUGGCUGAUACUAACUUG 629-647 AD-59624.1 A-120466.1GUGAUGACAUCACCAUGGU A-120467.1 ACCAUGGUGAUGUCAUCAC 1042-1060 AD-59625.1A-120482.1 UUCCAAGAAUGACAAUGAU A-120483.1 AUCAUUGUCAUUCUUGGAA 419-437AD-59626.1 A-120498.1 UGAUGGAGGUAUUUAAGUU A-120499.1 AACUUAAAUACCUCCAUCA520-538 AD-59627.1 A-120514.1 GUAUUCCAAUGUGAUAGGA A-120515.1UCCUAUCACAUUGGAAUAC 122-140 AD-59628.1 A-120530.1 CAGCCAAGCCGCGGGACAUA-120531.1 AUGUCCCGCGGCUUGGCUG 241-259 AD-59629.1 A-120452.1CAUGCCCCGCUUCCGCAUU A-120453.1 AAUGCGGAAGCGGGGCAUG 1172-1190 AD-59630.1A-120468.1 AUGUGAUAGGAACUGUAAC A-120469.1 GUUACAGUUCCUAUCACAU 130-148AD-59631.1 A-120484.1 ACAAAUCCCUUACCUUCAA A-120485.1 UUGAAGGUAAGGGAUUUGU661-679 AD-59632.1 A-120500.1 AGGUUUAUCUUUUGUCCUU A-120501.1AAGGACAAAAGAUAAACCU 163-181 AD-59633.1 A-120516.1 AGAUCCCGGAGGCCACCAAA-120517.1 UUGGUGGCCUCCGGGAUCU 331-349 AD-59634.1 A-120532.1ACAUUUUCCUGUCACCCCU A-120533.1 AGGGGUGACAGGAAAAUGU 439-457 AD-59635.1A-120548.1 GGCCUUUCCUGGUUUUUAU A-120549.1 AUAAAAACCAGGAAAGGCC 1432-1450AD-59636.1 A-120564.1 UUGUGUUAAGUAAAAUGUU A-120565.1 AACAUUUUACUUAACACAA1502-1520 AD-59637.1 A-120534.1 CAAGGAAAAUGCAGAGCAA A-120535.1UUGCUCUGCAUUUUCCUUG 740-758 AD-59638.1 A-120550.1 CUGAGAAAACAUCUGAUCAA-120551.1 UGAUCAGAUGUUUUCUCAG 550-568 AD-59639.1 A-120566.1UUACUUCAAGGGCCUGUGG A-120567.1 CCACAGGCCCUUGAAGUAA 872-890 AD-59640.1A-120536.1 ACCCCAACAGGGUGACUUU A-120537.1 AAAGUCACCCUGUUGGGGU 1402-1420AD-59641.1 A-120552.1 CCAGGUAUUGUUGCAGAAG A-120553.1 CUUCUGCAACAAUACCUGG1269-1287 AD-59642.1 A-120568.1 GCCUGUGGAAGUCAAAGUU A-120569.1AACUUUGACUUCCACAGGC 883-901 AD-59643.1 A-120538.1 UGGAACCUCUGCGAGAUUUA-120539.1 AAAUCUCGCAGAGGUUCCA 20-38 AD-59644.1 A-120554.1AGCCCUGAGAACACAAGGA A-120555.1 UCCUUGUGUUCUCAGGGCU 903-921 AD-59645.1A-120570.1 CUCUAUGUCUCAGAUGCAU A-120571.1 AUGCAUCUGAGACAUAGAG 1299-1317AD-59646.1 A-120540.1 AAGACCGAAGGCCGAAUCA A-120541.1 UGAUUCGGCCUUCGGUCUU792-810 AD-59647.1 A-120556.1 UAAAAUGUUCUUAUUCUUU A-120557.1AAAGAAUAAGAACAUUUUA 1512-1530 AD-59648.1 A-120572.1 UCUGGAAAAAGGAAGGUUUA-120573.1 AAACCUUCCUUUUUCCAGA 150-168 AD-59649.1 A-120542.1UUCAAGGCCAACAGGCCUU A-120543.1 AAGGCCUGUUGGCCUUGAA 1419-1437 AD-59650.1A-120558.1 GAAGUCAAAGUUCAGCCCU A-120559.1 AGGGCUGAACUUUGACUUC 890-908AD-59651.1 A-120574.1 CCAUCAAUGAGCUCACUGU A-120575.1 ACAGUGAGCUCAUUGAUGG832-850 AD-59652.1 A-120544.1 CUCUGAACACUAUUAUCUU A-120545.1AAGAUAAUAGUGUUCAGAG 1462-1480 AD-59653.1 A-120560.1 UGCUGGUUAACACCAUUUAA-120561.1 UAAAUGGUGUUAACCAGCA 856-874 AD-59654.1 A-120546.1AGAGGAAAGAACCAGUUUU A-120547.1 AAAACUGGUUCUUUCCUCU 39-57 AD-59655.1A-120562.1 UGCCCAGCCCUGUGGAAGA A-120563.1 UCUUCCACAGGGCUGGGCA  92-110

TABLE 21 AT3 (SERPINC1) modified sequences (The “Sense Sequence”column sequences are disclosed as SEQ ID NOS 1305-1467,respectively, in order of appearance, and the “Antisense Sequence”column sequences are disclosed asSEQ ID NOS 1468-1630, respectively, in order of appearance) Sense AntisDuplex Oligo Oligo Name Name oligoSeq Name oligoSeq AD-59267.1A-120250.1 GGAGAAGAAGGCAACUGAGdTdT A-120251.1 CUCAGUUGCCUUCUUCUCCdTdTAD-59268.1 A-120266.1 UGACCAAGCUGGGUGCCUGdTdT A-120267.1CAGGCACCCAGCUUGGUCAdTdT AD-59269.1 A-120282.1 GGUUAACACCAUUUACUUCdTdTA-120283.1 GAAGUAAAUGGUGUUAACCdTdT AD-59270.1 A-120298.1GCUGGUUAACACCAUUUACdTdT A-120299.1 GUAAAUGGUGUUAACCAGCdTdT AD-59271.1A-120314.1 UAAUGACACCCUCCAGCAAdTdT A-120315.1 UUGCUGGAGGGUGUCAUUAdTdTAD-59272.1 A-120330.1 CGUGUUCAGCAUCUAUGAUdTdT A-120331.1AUCAUAGAUGCUGAACACGdTdT AD-59273.1 A-120252.1 UUGAGGACGGCUUCAGUUUdTdTA-120253.1 AAACUGAAGCCGUCCUCAAdTdT AD-59274.1 A-120268.1CGGCGUGUCUGGGAACUGUdTdT A-120269.1 ACAGUUCCCAGACACGCCGdTdT AD-59275.1A-120284.1 UUAACACCAUUUACUUCAAdTdT A-120285.1 UUGAAGUAAAUGGUGUUAAdTdTAD-59276.1 A-120300.1 CCCUGAAAAGUCCAAACUCdTdT A-120301.1GAGUUUGGACUUUUCAGGGdTdT AD-59277.1 A-120316.1 CGAGAUGACCUCUAUGUCUdTdTA-120317.1 AGACAUAGAGGUCAUCUCGdTdT AD-59278.1 A-120332.1UCUACAAGGCUGAUGGAGAdTdT A-120333.1 UCUCCAUCAGCCUUGUAGAdTdT AD-59279.1A-120254.1 AGCUCACUGUUCUGGUGCUdTdT A-120255.1 AGCACCAGAACAGUGAGCUdTdTAD-59280.1 A-120270.1 AGGAGCAGCUGCAAGACAUdTdT A-120271.1AUGUCUUGCAGCUGCUCCUdTdT AD-59281.1 A-120286.1 GCCACCAACCGGCGUGUCUdTdTA-120287.1 AGACACGCCGGUUGGUGGCdTdT AD-59282.1 A-120302.1CAGAACAGAAGAUCCCGGAdTdT A-120303.1 UCCGGGAUCUUCUGUUCUGdTdT AD-59283.1A-120318.1 CCUUGUCGAUCUGUUCAGCdTdT A-120319.1 GCUGAACAGAUCGACAAGGdTdTAD-59284.1 A-120334.1 AGGCAAGUUCCGUUAUCGGdTdT A-120335.1CCGAUAACGGAACUUGCCUdTdT AD-59285.1 A-120256.1 UUUUGUCCUUGCUGCUCAUdTdTA-120257.1 AUGAGCAGCAAGGACAAAAdTdT AD-59286.1 A-120272.1AGACCUACCAGGACAUCAGdTdT A-120273.1 CUGAUGUCCUGGUAGGUCUdTdT AD-59287.1A-120288.1 AACUGAACUGCCGACUCUAdTdT A-120289.1 UAGAGUCGGCAGUUCAGUUdTdTAD-59288.1 A-120304.1 CAUUUACUUCAAGGGCCUGdTdT A-120305.1CAGGCCCUUGAAGUAAAUGdTdT AD-59289.1 A-120320.1 CCCUGGACUUCAAGGAAAAdTdTA-120321.1 UUUUCCUUGAAGUCCAGGGdTdT AD-59290.1 A-120336.1AGCUGCAAGUACCGCUGUUdTdT A-120337.1 AACAGCGGUACUUGCAGCUdTdT AD-59291.1A-120258.1 ACACAAGGAAGGAACUGUUdTdT A-120259.1 AACAGUUCCUUCCUUGUGUdTdTAD-59292.1 A-120274.1 GCAACUGAGGAUGAGGGCUdTdT A-120275.1AGCCCUCAUCCUCAGUUGCdTdT AD-59293.1 A-120290.1 GUAGCCAACCCUUGUGUUAdTdTA-120291.1 UAACACAAGGGUUGGCUACdTdT AD-59294.1 A-120306.1GUUUGUGAACAGAAGUAAAdTdT A-120307.1 UUUACUUCUGUUCACAAACdTdT AD-59295.1A-120322.1 GGGUGACUUUCAAGGCCAAdTdT A-120323.1 UUGGCCUUGAAAGUCACCCdTdTAD-59296.1 A-120338.1 UUAUCGGCGCGUGGCUGAAdTdT A-120339.1UUCAGCCACGCGCCGAUAAdTdT AD-59297.1 A-120260.1 CCACUUCUUCUUUGCCAAAdTdTA-120261.1 UUUGGCAAAGAAGAAGUGGdTdT AD-59298.1 A-120276.1AACACCAUUUACUUCAAGGdTdT A-120277.1 CCUUGAAGUAAAUGGUGUUdTdT AD-59299.1A-120292.1 GAUGGAGAGUCGUGUUCAGdTdT A-120293.1 CUGAACACGACUCUCCAUCdTdTAD-59300.1 A-120308.1 CACCAUUUACUUCAAGGGCdTdT A-120309.1GCCCUUGAAGUAAAUGGUGdTdT AD-59301.1 A-120324.1 UUUACUUCAAGGGCCUGUGdTdTA-120325.1 CACAGGCCCUUGAAGUAAAdTdT AD-59302.1 A-120340.1UAAGAGAAGUUCCUCUGAAdTdT A-120341.1 UUCAGAGGAACUUCUCUUAdTdT AD-59303.1A-120262.1 GCGGGACAUUCCCAUGAAUdTdT A-120263.1 AUUCAUGGGAAUGUCCCGCdTdTAD-59304.1 A-120278.1 UGCCCCACCCUGUCCUCUGdTdT A-120279.1CAGAGGACAGGGUGGGGCAdTdT AD-59305.1 A-120294.1 UGGUUAACACCAUUUACUUdTdTA-120295.1 AAGUAAAUGGUGUUAACCAdTdT AD-59306.1 A-120310.1CGGAUUGCCUCAGAUCACAdTdT A-120311.1 UGUGAUCUGAGGCAAUCCGdTdT AD-59307.1A-120326.1 CCAGGACAUCAGUGAGUUGdTdT A-120327.1 CAACUCACUGAUGUCCUGGdTdTAD-59308.1 A-120342.1 CCAGUUUUCAGGCGGAUUGdTdT A-120343.1CAAUCCGCCUGAAAACUGGdTdT AD-59309.1 A-120264.1 CACCAUAUCUGAGAAAACAdTdTA-120265.1 UGUUUUCUCAGAUAUGGUGdTdT AD-59310.1 A-120280.1AAGUAAAAAUAAAUACAAAdTdT A-120281.1 UUUGUAUUUAUUUUUACUUdTdT AD-59311.1A-120296.1 GCACCCAGGUGCUUGAGUUdTdT A-120297.1 AACUCAAGCACCUGGGUGCdTdTAD-59312.1 A-120312.1 UAACACCAUUUACUUCAAGdTdT A-120313.1CUUGAAGUAAAUGGUGUUAdTdT AD-59313.1 A-120328.1 CCUUCAAAGGUGAUGACAUdTdTA-120329.1 AUGUCAUCACCUUUGAAGGdTdT AD-59314.1 A-120344.1CAAGGCCAAUUCCCGCUUUdTdT A-120345.1 AAAGCGGGAAUUGGCCUUGdTdT AD-59315.1A-120360.1 UCAGUUUGAAGGAGCAGCUdTdT A-120361.1 AGCUGCUCCUUCAAACUGAdTdTAD-59316.1 A-120376.1 GGGACUGCGUGACCUGUCAdTdT A-120377.1UGACAGGUCACGCAGUCCCdTdT AD-59317.1 A-120392.1 UCAGCCAAUCGCCUUUUUGdTdTA-120393.1 CAAAAAGGCGAUUGGCUGAdTdT AD-59318.1 A-120408.1CCUCGGAAGCCAUCAAUGAdTdT A-120409.1 UCAUUGAUGGCUUCCGAGGdTdT AD-59319.1A-120424.1 GACAAUGAUAACAUUUUCCdTdT A-120425.1 GGAAAAUGUUAUCAUUGUCdTdTAD-59320.1 A-120346.1 CUUAUUCUUUGCACCUCUUdTdT A-120347.1AAGAGGUGCAAAGAAUAAGdTdT AD-59321.1 A-120362.1 AUUGCUGGCCGUUCGCUAAdTdTA-120363.1 UUAGCGAACGGCCAGCAAUdTdT AD-59322.1 A-120378.1AAAUGAAGAAGGCAGUGAAdTdT A-120379.1 UUCACUGCCUUCUUCAUUUdTdT AD-59323.1A-120394.1 UGAGUUGGUAUAUGGAGCCdTdT A-120395.1 GGCUCCAUAUACCAACUCAdTdTAD-59324.1 A-120410.1 UCCAGCAACUGAUGGAGGUdTdT A-120411.1ACCUCCAUCAGUUGCUGGAdTdT AD-59325.1 A-120426.1 AACUGUAACCUCUGGAAAAdTdTA-120427.1 UUUUCCAGAGGUUACAGUUdTdT AD-59326.1 A-120348.1UCAACAAAUGGGUGUCCAAdTdT A-120349.1 UUGGACACCCAUUUGUUGAdTdT AD-59327.1A-120364.1 GAUUAGCGGCCAUGUAUUCdTdT A-120365.1 GAAUACAUGGCCGCUAAUCdTdTAD-59328.1 A-120380.1 GUGCUUGAGUUGCCCUUCAdTdT A-120381.1UGAAGGGCAACUCAAGCACdTdT AD-59329.1 A-120396.1 UGCAGAAGGCCGAGAUGACdTdTA-120397.1 GUCAUCUCGGCCUUCUGCAdTdT AD-59330.1 A-120412.1CUAUUUUUGGUUUGUGAACdTdT A-120413.1 GUUCACAAACCAAAAAUAGdTdT AD-59331.1A-120428.1 CAGUGAAGCAGCUGCAAGUdTdT A-120429.1 ACUUGCAGCUGCUUCACUGdTdTAD-59332.1 A-120350.1 GUGUCCAAUAAGACCGAAGdTdT A-120351.1CUUCGGUCUUAUUGGACACdTdT AD-59333.1 A-120366.1 AUGAAUUGGAGGAGAUGAUdTdTA-120367.1 AUCAUCUCCUCCAAUUCAUdTdT AD-59334.1 A-120382.1AUCUAUGAUGUACCAGGAAdTdT A-120383.1 UUCCUGGUACAUCAUAGAUdTdT AD-59335.1A-120398.1 CCAUAAGGCAUUUCUUGAGdTdT A-120399.1 CUCAAGAAAUGCCUUAUGGdTdTAD-59336.1 A-120414.1 AUGCAUUCCAUAAGGCAUUdTdT A-120415.1AAUGCCUUAUGGAAUGCAUdTdT AD-59337.1 A-120430.1 UGGUGCUGGUUAACACCAUdTdTA-120431.1 AUGGUGUUAACCAGCACCAdTdT AD-59338.1 A-120352.1AUCUGUUCAGCCCUGAAAAdTdT A-120353.1 UUUUCAGGGCUGAACAGAUdTdT AD-59339.1A-120368.1 AGAUGAUGCUGGUGGUCCAdTdT A-120369.1 UGGACCACCAGCAUCAUCUdTdTAD-59340.1 A-120384.1 AUAUGGAGCCAAGCUCCAGdTdT A-120385.1CUGGAGCUUGGCUCCAUAUdTdT AD-59341.1 A-120400.1 CCGAAUCACCGAUGUCAUUdTdTA-120401.1 AAUGACAUCGGUGAUUCGGdTdT AD-59342.1 A-120416.1GCAGAGCAAUCCAGAGCGGdTdT A-120417.1 CCGCUCUGGAUUGCUCUGCdTdT AD-59343.1A-120432.1 ACACCAUUUACUUCAAGGGdTdT A-120433.1 CCCUUGAAGUAAAUGGUGUdTdTAD-59344.1 A-120354.1 GUACCAGGAAGGCAAGUUCdTdT A-120355.1GAACUUGCCUUCCUGGUACdTdT AD-59345.1 A-120370.1 UGCCCAAGCCUGAGAAGAGdTdTA-120371.1 CUCUUCUCAGGCUUGGGCAdTdT AD-59346.1 A-120386.1UUCUUUGCCAAACUGAACUdTdT A-120387.1 AGUUCAGUUUGGCAAAGAAdTdT AD-59347.1A-120402.1 UGGCCAAGGUAGAGAAGGAdTdT A-120403.1 UCCUUCUCUACCUUGGCCAdTdTAD-59348.1 A-120418.1 UGCUGCAAGAGUGGCUGGAdTdT A-120419.1UCCAGCCACUCUUGCAGCAdTdT AD-59349.1 A-120434.1 CCAUGUGCAUUUACCGCUCdTdTA-120435.1 GAGCGGUAAAUGCACAUGGdTdT AD-59350.1 A-120356.1AGACAUGGGCCUUGUCGAUdTdT A-120357.1 AUCGACAAGGCCCAUGUCUdTdT AD-59351.1A-120372.1 UUUUGGAGACAAAUCCCUUdTdT A-120373.1 AAGGGAUUUGUCUCCAAAAdTdTAD-59352.1 A-120388.1 GGAUGAGGGCUCAGAACAGdTdT A-120389.1CUGUUCUGAGCCCUCAUCCdTdT AD-59353.1 A-120404.1 CGGCUUUUGCUAUGACCAAdTdTA-120405.1 UUGGUCAUAGCAAAAGCCGdTdT AD-59354.1 A-120420.1AGCUCCAGCCCCUGGACUUdTdT A-120421.1 AAGUCCAGGGGCUGGAGCUdTdT AD-59355.1A-120436.1 CUGAUCAGAUCCACUUCUUdTdT A-120437.1 AAGAAGUGGAUCUGAUCAGdTdTAD-59356.1 A-120358.1 UGCUGGUGGUCCACAUGCCdTdT A-120359.1GGCAUGUGGACCACCAGCAdTdT AD-59357.1 A-120374.1 GCGAGAUUUAGAGGAAAGAdTdTA-120375.1 UCUUUCCUCUAAAUCUCGCdTdT AD-59358.1 A-120390.1CUGCUCAUUGGCUUCUGGGdTdT A-120391.1 CCCAGAAGCCAAUGAGCAGdTdT AD-59359.1A-120406.1 CCUUCAAUGAGACCUACCAdTdT A-120407.1 UGGUAGGUCUCAUUGAAGGdTdTAD-59360.1 A-120422.1 ACCAUUUACUUCAAGGGCCdTdT A-120423.1GGCCCUUGAAGUAAAUGGUdTdT AD-59587.1 A-120438.1 GCACCUCUUCCUAUUUUUGdTdTA-120439.1 CAAAAAUAGGAAGAGGUGCdTdT AD-59588.1 A-120454.1GUGGCUGAAGGCACCCAGGdTdT A-120455.1 CCUGGGUGCCUUCAGCCACdTdT AD-59589.1A-120470.1 UCCGCAUUGAGGACGGCUUdTdT A-120471.1 AAGCCGUCCUCAAUGCGGAdTdTAD-59590.1 A-120486.1 UGGACAUCUGCACAGCCAAdTdT A-120487.1UUGGCUGUGCAGAUGUCCAdTdT AD-59591.1 A-120502.1 GUCCAAACUCCCAGGUAUUdTdTA-120503.1 AAUACCUGGGAGUUUGGACdTdT AD-59592.1 A-120518.1AGAAGGAACUCACCCCAGAdTdT A-120519.1 UCUGGGGUGAGUUCCUUCUdTdT AD-59593.1A-120440.1 UCUUGAGGUAAAUGAAGAAdTdT A-120441.1 UUCUUCAUUUACCUCAAGAdTdTAD-59594.1 A-120456.1 AGCCCUGUGGACAUCUGCAdTdT A-120457.1UGCAGAUGUCCACAGGGCUdTdT AD-59595.1 A-120472.1 CAGAGCGGCCAUCAACAAAdTdTA-120473.1 UUUGUUGAUGGCCGCUCUGdTdT AD-59596.1 A-120488.1AUUUAAGUUUGACACCAUAdTdT A-120489.1 UAUGGUGUCAAACUUAAAUdTdT AD-59597.1A-120504.1 UGAGAAGAGCCUGGCCAAGdTdT A-120505.1 CUUGGCCAGGCUCUUCUCAdTdTAD-59598.1 A-120520.1 UCACCAUGGUCCUCAUCUUdTdT A-120521.1AAGAUGAGGACCAUGGUGAdTdT AD-59599.1 A-120442.1 GGAAGGAACUGUUCUACAAdTdTA-120443.1 UUGUAGAACAGUUCCUUCCdTdT AD-59600.1 A-120458.1CUGGUUUUUAUAAGAGAAGdTdT A-120459.1 CUUCUCUUAUAAAAACCAGdTdT AD-59601.1A-120474.1 CUGGGUGCCUGUAAUGACAdTdT A-120475.1 UGUCAUUACAGGCACCCAGdTdTAD-59602.1 A-120490.1 GUACCGCUGUUGUGAUUGCdTdT A-120491.1GCAAUCACAACAGCGGUACdTdT AD-59603.1 A-120506.1 UCUAUCAGCACCUGGCAGAdTdTA-120507.1 UCUGCCAGGUGCUGAUAGAdTdT AD-59604.1 A-120522.1CUGGCAGAUUCCAAGAAUGdTdT A-120523.1 CAUUCUUGGAAUCUGCCAGdTdT AD-59605.1A-120444.1 CGAUGUCAUUCCCUCGGAAdTdT A-120445.1 UUCCGAGGGAAUGACAUCGdTdTAD-59606.1 A-120460.1 GCUUCUGGGACUGCGUGACdTdT A-120461.1GUCACGCAGUCCCAGAAGCdTdT AD-59607.1 A-120476.1 CCUGUCACGGGAGCCCUGUdTdTA-120477.1 ACAGGGCUCCCGUGACAGGdTdT AD-59608.1 A-120492.1AUUUACUUCAAGGGCCUGUdTdT A-120493.1 ACAGGCCCUUGAAGUAAAUdTdT AD-59609.1A-120508.1 UGCUACCACUUUCUAUCAGdTdT A-120509.1 CUGAUAGAAAGUGGUAGCAdTdTAD-59610.1 A-120524.1 GGAACUGUCCAAGGCCAAUdTdT A-120525.1AUUGGCCUUGGACAGUUCCdTdT AD-59611.1 A-120446.1 ACAAAUCCUCCAAGUUAGUdTdTA-120447.1 ACUAACUUGGAGGAUUUGUdTdT AD-59612.1 A-120462.1AUUUACCGCUCCCCGGAGAdTdT A-120463.1 UCUCCGGGGAGCGGUAAAUdTdT AD-59613.1A-120478.1 ACCCCUGAGUAUCUCCACGdTdT A-120479.1 CGUGGAGAUACUCAGGGGUdTdTAD-59614.1 A-120494.1 CACUAUCUCCACUUGCCCAdTdT A-120495.1UGGGCAAGUGGAGAUAGUGdTdT AD-59615.1 A-120510.1 AAAUACAAACUACUUCCAUdTdTA-120511.1 AUGGAAGUAGUUUGUAUUUdTdT AD-59616.1 A-120526.1CUGGUUAACACCAUUUACUdTdT A-120527.1 AGUAAAUGGUGUUAACCAGdTdT AD-59617.1A-120448.1 UCAUCUUGCCCAAGCCUGAdTdT A-120449.1 UCAGGCUUGGGCAAGAUGAdTdTAD-59618.1 A-120464.1 CCUCAGAUCACACUAUCUCdTdT A-120465.1GAGAUAGUGUGAUCUGAGGdTdT AD-59619.1 A-120480.1 CUGUCCUCUGGAACCUCUGdTdTA-120481.1 CAGAGGUUCCAGAGGACAGdTdT AD-59620.1 A-120496.1CCCUGUGGAAGAUUAGCGGdTdT A-120497.1 CCGCUAAUCUUCCACAGGGdTdT AD-59621.1A-120512.1 AUCUCCACGGCUUUUGCUAdTdT A-120513.1 UAGCAAAAGCCGUGGAGAUdTdTAD-59622.1 A-120528.1 GUGGCUGGAUGAAUUGGAGdTdT A-120529.1CUCCAAUUCAUCCAGCCACdTdT AD-59623.1 A-120450.1 CAAGUUAGUAUCAGCCAAUdTdTA-120451.1 AUUGGCUGAUACUAACUUGdTdT AD-59624.1 A-120466.1GUGAUGACAUCACCAUGGUdTdT A-120467.1 ACCAUGGUGAUGUCAUCACdTdT AD-59625.1A-120482.1 UUCCAAGAAUGACAAUGAUdTdT A-120483.1 AUCAUUGUCAUUCUUGGAAdTdTAD-59626.1 A-120498.1 UGAUGGAGGUAUUUAAGUUdTdT A-120499.1AACUUAAAUACCUCCAUCAdTdT AD-59627.1 A-120514.1 GUAUUCCAAUGUGAUAGGAdTdTA-120515.1 UCCUAUCACAUUGGAAUACdTdT AD-59628.1 A-120530.1CAGCCAAGCCGCGGGACAUdTdT A-120531.1 AUGUCCCGCGGCUUGGCUGdTdT AD-59629.1A-120452.1 CAUGCCCCGCUUCCGCAUUdTdT A-120453.1 AAUGCGGAAGCGGGGCAUGdTdTAD-59630.1 A-120468.1 AUGUGAUAGGAACUGUAACdTdT A-120469.1GUUACAGUUCCUAUCACAUdTdT AD-59631.1 A-120484.1 ACAAAUCCCUUACCUUCAAdTdTA-120485.1 UUGAAGGUAAGGGAUUUGUdTdT AD-59632.1 A-120500.1AGGUUUAUCUUUUGUCCUUdTdT A-120501.1 AAGGACAAAAGAUAAACCUdTdT AD-59633.1A-120516.1 AGAUCCCGGAGGCCACCAAdTdT A-120517.1 UUGGUGGCCUCCGGGAUCUdTdTAD-59634.1 A-120532.1 ACAUUUUCCUGUCACCCCUdTdT A-120533.1AGGGGUGACAGGAAAAUGUdTdT AD-59635.1 A-120548.1 GGCCUUUCCUGGUUUUUAUdTdTA-120549.1 AUAAAAACCAGGAAAGGCCdTdT AD-59636.1 A-120564.1UUGUGUUAAGUAAAAUGUUdTdT A-120565.1 AACAUUUUACUUAACACAAdTdT AD-59637.1A-120534.1 CAAGGAAAAUGCAGAGCAAdTdT A-120535.1 UUGCUCUGCAUUUUCCUUGdTdTAD-59638.1 A-120550.1 CUGAGAAAACAUCUGAUCAdTdT A-120551.1UGAUCAGAUGUUUUCUCAGdTdT AD-59639.1 A-120566.1 UUACUUCAAGGGCCUGUGGdTdTA-120567.1 CCACAGGCCCUUGAAGUAAdTdT AD-59640.1 A-120536.1ACCCCAACAGGGUGACUUUdTdT A-120537.1 AAAGUCACCCUGUUGGGGUdTdT AD-59641.1A-120552.1 CCAGGUAUUGUUGCAGAAGdTdT A-120553.1 CUUCUGCAACAAUACCUGGdTdTAD-59642.1 A-120568.1 GCCUGUGGAAGUCAAAGUUdTdT A-120569.1AACUUUGACUUCCACAGGCdTdT AD-59643.1 A-120538.1 UGGAACCUCUGCGAGAUUUdTdTA-120539.1 AAAUCUCGCAGAGGUUCCAdTdT AD-59644.1 A-120554.1AGCCCUGAGAACACAAGGAdTdT A-120555.1 UCCUUGUGUUCUCAGGGCUdTdT AD-59645.1A-120570.1 CUCUAUGUCUCAGAUGCAUdTdT A-120571.1 AUGCAUCUGAGACAUAGAGdTdTAD-59646.1 A-120540.1 AAGACCGAAGGCCGAAUCAdTdT A-120541.1UGAUUCGGCCUUCGGUCUUdTdT AD-59647.1 A-120556.1 UAAAAUGUUCUUAUUCUUUdTdTA-120557.1 AAAGAAUAAGAACAUUUUAdTdT AD-59648.1 A-120572.1UCUGGAAAAAGGAAGGUUUdTdT A-120573.1 AAACCUUCCUUUUUCCAGAdTdT AD-59649.1A-120542.1 UUCAAGGCCAACAGGCCUUdTdT A-120543.1 AAGGCCUGUUGGCCUUGAAdTdTAD-59650.1 A-120558.1 GAAGUCAAAGUUCAGCCCUdTdT A-120559.1AGGGCUGAACUUUGACUUCdTdT AD-59651.1 A-120574.1 CCAUCAAUGAGCUCACUGUdTdTA-120575.1 ACAGUGAGCUCAUUGAUGGdTdT AD-59652.1 A-120544.1CUCUGAACACUAUUAUCUUdTdT A-120545.1 AAGAUAAUAGUGUUCAGAGdTdT AD-59653.1A-120560.1 UGCUGGUUAACACCAUUUAdTdT A-120561.1 UAAAUGGUGUUAACCAGCAdTdTAD-59654.1 A-120546.1 AGAGGAAAGAACCAGUUUUdTdT A-120547.1AAAACUGGUUCUUUCCUCUdTdT AD-59655.1 A-120562.1 UGCCCAGCCCUGUGGAAGAdTdTA-120563.1 UCUUCCACAGGGCUGGGCAdTdT

Cell Culture and Transfections

HepG2 cells (ATCC, Manassas, Va.) were grown to near confluence at 37°C. in an atmosphere of 5% CO2 in Eagle's Minimum Essential Medium (ATCC)supplemented with 10% FBS, streptomycin, and glutamine (ATCC) beforebeing released from the plate by trypsinization. Transfection wascarried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of LipofectamineRNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl ofeach of the 164 siRNA duplexes to an individual well in a 96-well plate.The mixture was then incubated at room temperature for 15 minutes. 80 μlof complete growth media without antibiotic containing ˜2.5×10⁴ HepG2cells were then added to the siRNA mixture. Cells were incubated for 24hours prior to RNA purification. Experiments were performed at 20 nM andincluded naïve cells and cells transfected with AD-1955, a luciferasetargeting siRNA as negative controls.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12)

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minute at 700 rpm on a platform shaker (the mixing speed wasthe same throughout the process). Ten microliters of magnetic beads and80 μl Lysis/Binding Buffer mixture were added to a round bottom plateand mixed for 1 minute. Magnetic beads were captured using magneticstand and the supernatant was removed without disturbing the beads.After removing supernatant, the lysed cells were added to the remainingbeads and mixed for 5 minutes. After removing supernatant, magneticbeads were washed 2 times with 150 μl Wash Buffer A and mixed for 1minute. Beads were capture again and supernatant removed. Beads werethen washed with 150 μl Wash Buffer B, captured and supernatant wasremoved. Beads were next washed with 150 μl Elution Buffer, captured andsupernatant removed. Beads were allowed to dry for 2 minutes. Afterdrying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70°C. Beads were captured on magnet for 5 minutes. Forty 111 ofsupernatant, containing the isolated RNA was removed and added toanother 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O perreaction were added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR

Two μl of cDNA were added to a master mix containing 0.5 μl human GAPDHTaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl human SERPINC1TaqMan probe (Applied Biosystems cat # Hs00892758_m1) and 5 μlLightcycler 480 probe master mix (Roche Cat #04887301001) per well in a384-well plate (Roche cat #04887301001). Real time PCR was done in anLC480 Real Time PCR machine (Roche).

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 20 nM AD-1955.

Table 22 shows the results of a single dose screen in HepG2 of theindicated iRNAs.

TABLE 22² 20 nM single dose screen of the indicated iRNAs 20 nM StandardDuplex ID Average Deviation AD1955 100.4 9.1 AD-59267.1 9.3 2.7AD-59268.1 72.2 20.3 AD-59269.1 6.9 0.4 AD-59270.1 18.3 5.2 AD-59271.127.9 7.6 AD-59272.1 13.0 1.1 AD-59273.1 79.4 10.9 AD-59274.1 5.9 1.1AD-59275.1 16.1 5.5 AD-59276.1 6.1 2.2 AD-59277.1 4.4 0.9 AD-59278.1 9.30.4 AD-59279.1 12.7 4.7 AD-59280.1 4.5 1.7 AD-59281.1 15.7 5.6AD-59282.1 25.9 6.9 AD-59283.1 27.0 18.9 AD-59284.1 8.6 3.7 AD-59285.111.2 3.7 AD-59286.1 15.2 5.0 AD-59287.1 6.9 1.9 AD-59288.1 74.5 16.5AD-59289.1 3.3 1.3 AD-59290.1 13.8 2.5 AD-59291.1 9.4 2.8 AD-59292.1 9.53.5 AD-59293.1 2.5 1.1 AD-59294.1 4.8 1.8 AD-59295.1 11.8 7.2 AD-59296.132.4 4.9 AD-59297.1 78.5 105.0 AD-59298.1 76.3 10.7 AD-59299.1 4.4 0.8AD-59300.1 32.2 10.8 AD-59301.1 48.5 15.2 AD-59302.1 7.2 3.0 AD-59303.117.0 2.3 AD-59304.1 87.1 16.4 AD-59305.1 4.4 0.9 AD-59306.1 35.7 10.6AD-59307.1 6.3 0.4 AD-59308.1 65.1 9.1 AD-59309.1 7.5 2.0 AD-59310.127.1 9.5 AD-59311.1 8.1 1.4 AD-59312.1 84.5 8.5 AD-59313.1 17.8 0.2AD-59314.1 21.1 0.4 AD-59315.1 85.5 29.8 AD-59316.1 13.0 1.6 AD-59317.164.0 10.7 AD-59318.1 7.9 2.2 AD-59319.1 31.8 4.7 AD-59320.1 5.7 2.1AD-59321.1 3.4 0.0 AD-59322.1 9.6 1.3 AD-59323.1 100.1 4.8 AD-59324.140.2 2.9 AD-59325.1 5.8 0.7 AD-59326.1 20.4 10.9 AD-59327.1 5.0 2.0AD-59328.1 8.0 2.8 AD-59329.1 54.1 5.9 AD-59330.1 21.6 12.3 AD-59331.14.3 2.4 AD-59332.1 12.9 3.8 AD-59333.1 26.1 1.0 AD-59334.1 41.9 4.7AD-59335.1 12.5 1.7 AD-59336.1 13.5 1.7 AD-59337.1 78.6 3.6 AD-59338.117.9 12.3 AD-59339.1 5.8 4.1 AD-59340.1 92.3 10.0 AD-59341.1 8.0 1.8AD-59342.1 11.1 1.9 AD-59343.1 43.6 4.2 AD-59344.1 6.0 2.3 AD-59345.123.6 3.6 AD-59346.1 41.0 3.2 AD-59347.1 12.2 1.0 AD-59348.1 30.0 5.1AD-59349.1 14.4 0.9 AD-59350.1 9.1 1.0 AD-59351.1 10.7 1.6 AD-59352.11.9 0.4 AD-59353.1 4.6 0.8 AD-59354.1 30.1 0.1 AD-59355.1 12.2 2.2AD-59356.1 63.6 11.5 AD-59357.1 112.1 20.6 AD-59358.1 23.2 3.0AD-59359.1 7.5 0.8 AD-59360.1 19.2 0.5 AD-59587.1 8.4 2.7 AD-59588.124.8 5.7 AD-59589.1 4.4 1.7 AD-59590.1 13.6 1.3 AD-59591.1 3.1 0.5AD-59592.1 12.1 2.7 AD-59593.1 7.6 3.5 AD-59594.1 7.5 1.6 AD-59595.120.1 2.9 AD-59596.1 7.3 1.3 AD-59597.1 61.3 6.8 AD-59598.1 24.4 1.6AD-59599.1 4.3 0.4 AD-59600.1 30.1 2.8 AD-59601.1 11.4 0.8 AD-59602.16.0 0.0 AD-59603.1 19.4 2.2 AD-59604.1 5.8 0.6 AD-59605.1 6.5 0.3AD-59606.1 9.9 0.6 AD-59607.1 28.0 2.8 AD-59608.1 66.1 8.0 AD-59609.184.2 9.0 AD-59610.1 8.0 1.7 AD-59611.1 6.6 1.5 AD-59612.1 31.5 2.7AD-59613.1 27.6 1.7 AD-59614.1 20.3 0.5 AD-59615.1 12.8 1.4 AD-59616.16.8 0.3 AD-59617.1 13.1 0.9 AD-59618.1 5.4 0.2 AD-59619.1 59.1 5.4AD-59620.1 6.3 1.7 AD-59621.1 18.4 1.8 AD-59622.1 9.6 1.2 AD-59623.1 8.72.1 AD-59624.1 13.6 0.7 AD-59625.1 12.0 1.5 AD-59626.1 14.6 1.8AD-59627.1 7.7 6.7 AD-59628.1 7.6 0.3 AD-59629.1 55.7 8.2 AD-59630.120.2 5.9 AD-59631.1 12.3 0.1 AD-59632.1 3.1 0.3 AD-59633.1 20.4 2.1AD-59634.1 3.7 0.0 AD-59635.1 16.1 1.3 AD-59636.1 13.0 1.0 AD-59637.114.4 4.8 AD-59638.1 7.7 1.0 AD-59639.1 70.4 5.3 AD-59640.1 2.5 0.1AD-59641.1 18.1 2.1 AD-59642.1 5.9 1.0 AD-59643.1 70.7 17.8 AD-59644.117.9 4.7 AD-59645.1 2.5 0.1 AD-59646.1 19.9 0.1 AD-59647.1 74.8 12.0AD-59648.1 6.8 0.0 AD-59649.1 95.2 6.2 AD-59650.1 71.1 1.0 AD-59651.15.0 0.8 AD-59652.1 5.5 1.2 AD-59653.1 15.3 1.0 AD-59654.1 67.4 0.9AD-59655.1 10.8 1.0 Naive 94.9 14.2 ²Modified.

We claim:
 1. A double-stranded ribonucleic acid (dsRNA) for inhibitingexpression of Serpinc1, wherein said dsRNA comprises a sense strand andan antisense strand, wherein said sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of SEQ ID NO:1 and said antisense strand comprisesat least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of SEQ ID NO:5.
 2. Adouble-stranded ribonucleic acid (dsRNA) for inhibiting expression ofSerpinc1, wherein said dsRNA comprises a sense strand and an antisensestrand, the antisense strand comprising a region of complementaritywhich comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from any one of the antisense sequences listed in anyone of Tables 3, 4, 8, 11, 12, 14, 15, 20, and
 21. 3. The dsRNA of claim1 or 2, wherein said dsRNA comprises at least one modified nucleotide.4. The dsRNA of claim 1 or 2, wherein the dsRNA comprises a sense strandconsisting of a sense strand sequence selected from the sequence of anyone of Tables 3, 4, 8, 11, 12, 14, 15, 20, and 21, and an antisensestrand consisting of an antisense sequence selected from the sequencesof any one of Tables 3, 4, 8, 11, 12, 14, 15, 20, and
 21. 5. A cellcontaining the dsRNA of claim 1 or
 2. 6. A vector encoding at least onestrand of a dsRNA, wherein said dsRNA comprises a region ofcomplementarity to at least a part of an mRNA encoding Serpinc1, whereinsaid dsRNA is 30 base pairs or less in length, and wherein said dsRNAtargets said mRNA for cleavage.
 7. A pharmaceutical composition forinhibiting expression of a Serpinc1 gene comprising the dsRNA of claim 1or 2 or the vector of claim
 6. 8. A method of inhibiting Serpinc1expression in a cell, the method comprising: (a) contacting the cellwith the dsRNA of claim 1 or 2 or the vector of claim 6; and (b)maintaining the cell produced in step (a) for a time sufficient toobtain degradation of the mRNA transcript of a Serpinc1 gene, therebyinhibiting expression of the Serpinc1 gene in the cell.
 9. A method oftreating a subject having a disorder that would benefit from reductionin Serpinc1 expression, comprising administering to the subject atherapeutically effective amount of the dsRNA of claim 1 or 2 or thevector of claim 6 thereby treating said subject.
 10. A method ofpreventing at least one symptom in a subject having a disorder thatwould benefit from reduction in Serpinc1 expression, comprisingadministering to the subject a therapeutically effective amount of thedsRNA of claim 1 or 2 or the vector of claim 6, thereby preventing atleast one symptom in the subject having a disorder that would benefitfrom reduction in Serpinc1 expression.
 11. The method of claim 9 or 10,wherein the disorder is a bleeding disorder.
 12. The method of claim 11,wherein the bleeding disorder is a hemophilia.
 13. A method ofinhibiting the expression of Serpinc1 in a subject, the methodcomprising administering to said subject a therapeutically effectiveamount of the dsRNA of claim 1 or 2 or the vector of claim 6, therebyinhibiting the expression of Serpinc1 in said subject.